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ArF Excimer Laser Corneal Ablation

Permanent Link: http://ufdc.ufl.edu/UFE0022631/00001

Material Information

Title: ArF Excimer Laser Corneal Ablation Effects of Laser Repetition Rate and Fundamental Laser-Tissue Coupling
Physical Description: 1 online resource (161 p.)
Language: english
Creator: Shanyfelt, Leia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ablation, cornea, histology, imaging, interferometry, lasik, photoablation, plume, prk, spectrometry, threshold, transmission
Mechanical and Aerospace Engineering -- Dissertations, Academic -- UF
Genre: Mechanical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Several topics in excimer laser corneal ablation remain unresolved, ranging from fundamental to practical. The roles that photothermal and photochemical processes play in the mechanism of corneal ablation remain a topic of research, including laser-tissue coupling below the ablation threshold. Goals of the present work are to investigate the mechanism of photoablation and to assess whether bovine corneal ablations generated at laser repetition rates up to 400 Hz are comparable to ablations performed at current clinical rates (60?100 Hz). A combination of experiments was implemented, including ablation plume dynamics, corneal ablation profiles and high-resolution microscopy. Using white-light interferometry analysis, no statistical difference was found between corneal ablation profiles created at 60 Hz and 400 Hz. Using plume imaging and transmission studies, the bulk ablation plume was found to dissipate on a time-scale less than the pulse-to-pulse separation for repetition rates up to about 400 Hz. A persistent, diffuse component of the ablation products was observed to be comparable at both rates. Microscopy did not reveal signs of thermal tissue damage for repetition rates up to 400 Hz. Ablations performed on PMMA did not reveal repetition rate effects. Ablation pattern algorithm reversal and plume extractor addition were analyzed for potential effects on the clinical outcome. Increasing laser repetition rates for clinical applications appear feasible. Sub-ablative fluences utilizing 193-nm and 355-nm perturbations yielded insight into photochemistry of collagen and amino acids. Amino acid solutions were not permanently altered by either wavelength. For collagen solutions, an average of 28 photons at 193 nm was required to break a peptide bond. 355-nm perturbations resulted in an average of 508 photons required to break a peptide bond. A dynamic photobleaching occurs in both amino acid and collagen solutions at both wavelengths and is greater at 193 nm than at 355 nm, resolving by more than tens of nanoseconds but less than tens of seconds. Permanent changes induced in the collagen samples are due to scission of peptide bond. Mass spectrometry experiments analyzed the ablation products in the ablation plume. The experiments indicate a primarily photochemical ablation mechanism with peptide bonds being the primary chromophore.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Leia Shanyfelt.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Hahn, David W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022631:00001

Permanent Link: http://ufdc.ufl.edu/UFE0022631/00001

Material Information

Title: ArF Excimer Laser Corneal Ablation Effects of Laser Repetition Rate and Fundamental Laser-Tissue Coupling
Physical Description: 1 online resource (161 p.)
Language: english
Creator: Shanyfelt, Leia
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2008

Subjects

Subjects / Keywords: ablation, cornea, histology, imaging, interferometry, lasik, photoablation, plume, prk, spectrometry, threshold, transmission
Mechanical and Aerospace Engineering -- Dissertations, Academic -- UF
Genre: Mechanical Engineering thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Several topics in excimer laser corneal ablation remain unresolved, ranging from fundamental to practical. The roles that photothermal and photochemical processes play in the mechanism of corneal ablation remain a topic of research, including laser-tissue coupling below the ablation threshold. Goals of the present work are to investigate the mechanism of photoablation and to assess whether bovine corneal ablations generated at laser repetition rates up to 400 Hz are comparable to ablations performed at current clinical rates (60?100 Hz). A combination of experiments was implemented, including ablation plume dynamics, corneal ablation profiles and high-resolution microscopy. Using white-light interferometry analysis, no statistical difference was found between corneal ablation profiles created at 60 Hz and 400 Hz. Using plume imaging and transmission studies, the bulk ablation plume was found to dissipate on a time-scale less than the pulse-to-pulse separation for repetition rates up to about 400 Hz. A persistent, diffuse component of the ablation products was observed to be comparable at both rates. Microscopy did not reveal signs of thermal tissue damage for repetition rates up to 400 Hz. Ablations performed on PMMA did not reveal repetition rate effects. Ablation pattern algorithm reversal and plume extractor addition were analyzed for potential effects on the clinical outcome. Increasing laser repetition rates for clinical applications appear feasible. Sub-ablative fluences utilizing 193-nm and 355-nm perturbations yielded insight into photochemistry of collagen and amino acids. Amino acid solutions were not permanently altered by either wavelength. For collagen solutions, an average of 28 photons at 193 nm was required to break a peptide bond. 355-nm perturbations resulted in an average of 508 photons required to break a peptide bond. A dynamic photobleaching occurs in both amino acid and collagen solutions at both wavelengths and is greater at 193 nm than at 355 nm, resolving by more than tens of nanoseconds but less than tens of seconds. Permanent changes induced in the collagen samples are due to scission of peptide bond. Mass spectrometry experiments analyzed the ablation products in the ablation plume. The experiments indicate a primarily photochemical ablation mechanism with peptide bonds being the primary chromophore.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Leia Shanyfelt.
Thesis: Thesis (Ph.D.)--University of Florida, 2008.
Local: Adviser: Hahn, David W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2009-02-28

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2008
System ID: UFE0022631:00001


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1 ArF EXCIMER LASER CORNEAL ABLATION: EFFECTS OF LASER REPETITION RATE AND FUNDAMENTAL LASE R-TISSUE COUPLING By LEIA MEGAN SHANYFELT A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008

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2 2008 Leia Megan Shanyfelt

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3 To my husband Dan Shanyfelt

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4 ACKNOWLEDGMENTS First and foremost, I thank my husband for maki ng my transition back to the University of Florida possible. Mostly, I thank him for his pa tience and support during less than ideal living circumstances. I am also grateful to my pa rents for always insist ing upon excellence in my academic career. I am thankful to Dr. Hahn for allowing me to return to his laborat ory to obtain my PhD under his advisement. I am appreciative to all of my lab mates for their assistance and friendship over the last few years. So thanks go out to Soupy Dalyander, Prasoon Diwaker, Phil Jackson, Bret Windom and Cary Henry. I would like to sp ecifically thank Cary Henry for his help in harvesting bovine eyes, a less than pleasurable task. Also, I am obliged to Soupy Dalyander for all of her Matlab help and to Prasoon Diwake r for his patient help with the imaging study. Additionally, I am appreciative to Dr. Pam Di ckrell for her many hours of assistance with the white-light interferometry measurements and to Dr. Greg Sawyer for the use of his interferometer and mass spectrometer. I am also indebted to Dr. Hank Edel hauser for his creation of the microscopy images. Also thanks go out to Dr. George Pettit and Alcon for supporting this research with resources, al gorithms, discussion and input.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4LIST OF TABLES................................................................................................................. ..........7LIST OF FIGURES................................................................................................................ .........9LIST OF ABBREVIATIONS........................................................................................................14ABSTRACT....................................................................................................................... ............15 CHAPTER 1 BACKGROUND....................................................................................................................17Excimer Laser Refractive Surgery..........................................................................................17Human Cornea.................................................................................................................17Clinical Implementation..................................................................................................21Clinical Issues................................................................................................................ ..22Polymer Ablation............................................................................................................... .....24Corneal Ablation............................................................................................................... ......28Experimental Methods and Studies........................................................................................31Plume Dynamics..............................................................................................................31White-Light Interferometry.............................................................................................33Mass Spectrocsopy..........................................................................................................35Objectives of the Present Work..............................................................................................372 PLUME DYNAMICS............................................................................................................43Experimental Setup and Methods...........................................................................................43Plume Imaging.................................................................................................................44Plume Transmission........................................................................................................44Plume Dynamics Results........................................................................................................46Plume Imaging.................................................................................................................46Plume Transmission........................................................................................................47Plume Dynamics Summary....................................................................................................503 ABLATION PROFILES........................................................................................................57Experimental Setup and Methods...........................................................................................57Wax Ablation Profiles.....................................................................................................57Plastic Ablation Profiles..................................................................................................60Imaged Ablation Profiles.................................................................................................61Ablation Profile Results....................................................................................................... ...62Wax Ablation Profiles.....................................................................................................62

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6 Imaged Ablation Profiles.................................................................................................68Ablation Profile Summary......................................................................................................704 HISTOLOGY...................................................................................................................... ...93Experimental Setup and Methods...........................................................................................93Histology Results.............................................................................................................. ......94Histology Summary.............................................................................................................. ..965 SUB-ABLATIVE PERTUBATIONS..................................................................................100Experimental Setup and Methods.........................................................................................100Sub-Ablative Results........................................................................................................... .105193-nm Perturbations....................................................................................................105355-nm Perturbations....................................................................................................111Sub-Ablative Summary........................................................................................................1136 MASS SPECTROSCOPY....................................................................................................132Experimental Setup and Methods.........................................................................................132Mass Spectrometry Results...................................................................................................133Mass Spectrometry Summary...............................................................................................1347 CONCLUSIONS AND FUTURE WORK...........................................................................141Conclusions.................................................................................................................... .......141Future Work.................................................................................................................... ......145 APPENDIX A MATLAB EDGE FINDER..................................................................................................147B MATLAB RESULTS COMPILER......................................................................................150C EQUIPMENT LISTING.......................................................................................................153LIST OF REFERENCES.............................................................................................................154BIOGRAPHICAL SKETCH.......................................................................................................161

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7 LIST OF TABLES Table page 1-1 Selected molecular bonds in tissu e and their dissociation energies (eV).........................413-1 Overall ablation depths ( m) for bovine straight-bore ablations for each interferogram for various laser repetition rates..................................................................773-2 Overall ablation depths ( m) for bovine straight-bore ablations for each laser repetition rates............................................................................................................... .....783-3 Overall ablation depths ( m) and standard deviations for bovine scanning ablations for each interferogram for vari ous laser repetition rates....................................................783-4 Overall ablation depths ( m) for bovine scanning ablations for each laser repetition rates.......................................................................................................................... ..........793-5 PMMA ablation depths ( m) and standard deviations ( m) for the stretched spiral and reverse spiral algorithms a nd ablation rates of 60, 92 and 400 Hz.............................853-6 Maximum ablation depth for paired st retched and reverse spiral ablation........................915-1 Average absorbance values and standard deviations for each pulse energy and time step with 193-nm perturbation of collagen solutions.......................................................1175-2 Average absorbance values and standard deviations for each pulse energy and time step with 193-nm perturba tion of amino acid solutions...................................................1185-3 Average extinction coefficient (cm-1) values and standard deviations for each pulse energy and time step with 193-nm perturbation of collagen solutions............................1195-4 Average extinction coefficient (cm-1) values and standard deviations for each pulse energy and time step with 193-nm pe rturbation of amino acid solutions........................1205-5 Average bond number densities (pep tide bonds/ml) and numbers of absorbers (peptide bonds) and standard deviations for each pulse energy and time step with 193-nm perturbation of collagen solutions......................................................................1235-6 Number of photons required to break a peptide bond for each pulse energy with 193nm perturbation of collagen solutions..............................................................................1265-7 Average number densities (peptide b onds/ml) and numbers of absorbers (peptide bonds) and standard deviations for each pul se energy and time step with 193-nm perturbation of collagen solutions....................................................................................1275-8 Average absorbance values and standard deviations for each pulse energy and time step with 355-nm perturbation of collagen solutions.......................................................129

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8 5-9 Average extinction coefficients and sta ndard deviations for each pulse energy and time step with 355-nm perturbation of collagen solutions...............................................130C-1 Manufacturers and Model numbers of experimental components...................................153

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9 LIST OF FIGURES Figure page 1-1 Schematic of the human eye..............................................................................................381-2 The five layers of the human cornea..................................................................................381-3 Schematic of the triple-h elix structure of collagen............................................................391-4 Simplified collagen struct ure of the human cornea...........................................................391-5 Ablation rate (microns/shot) ve rses Fluence (mJ/cm2) summary.....................................401-6 PMMA molecule.............................................................................................................. ..401-7 Semi-log plot of etch depth verses fluence in PMMA at 193 nm......................................411-8 A typical transmissi on experiment setup...........................................................................421-9 The setup of a traditiona l Michelson interferometer..........................................................422-1 Experimental set-up for m easuring ablation plume dynamics...........................................522-2 Schematic of the transmission studies setup......................................................................522-3 Ablation plume images as a function of time following the ablating laser pulse..............532-4 Long time scale ablation plume images.............................................................................542-5 The average scattering intensity (full-im age) of the ablation plume images as a function of time following the ablating laser pulse...........................................................542-6 ArF probe laser transmission through th e ablation plume as a function of time following ablation............................................................................................................. .552-7 Transmission of ArF probe b eam through non-ablating beam path..................................552-8 Configuration of the nitrogen purge jet for the transmission loss experiment..................562-9 Average transmission of the ArF probe la ser at a fixed delay of 1.25 ms and fixed height of ~2 mm................................................................................................................ .563-1 Experimental set-up for ablation of bovine corneas..........................................................713-2 White light interferometry 3-dimensional profile of a wax impr ession of a straightbore ablation crater........................................................................................................... .71

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10 3-3 White light interferometry 3-dimensiona l profile of a wax impression of a scanning ablation crater................................................................................................................ .....723-4 White light interferometry 2-dimensional profile of a wax impr ession of a straightbore crater.................................................................................................................... ......723-5 White light interferometry 2-dimensiona l cross sections of a wax impression of a scanning crater................................................................................................................ ...733-6 White light interferometry 2-dimensiona l profile of a wax impression of a scanning crater......................................................................................................................... .........733-7 White light interferometry 2-dimensiona l cross sections of a wax impression of a scanning crater................................................................................................................ ...743-8 White light interferometry 3-dimensional profile of a straightbore ablation crater on PMMA........................................................................................................................... ....743-9 White light interferometry 2-dimensional cross sections of a straight-bore ablation crater on PMMA................................................................................................................753-10 White light interferometry 3-dimensiona l profile of a scanning ablation crater on PMMA........................................................................................................................... ....753-11 White light interferometry 2-dimensional cross sections of a s canning ablation crater on PMMA........................................................................................................................ ..763-12 Experimental set-up for im aging ablation plume profiles.................................................763-13 Representative profile image of a bovine eye....................................................................773-14 Straight-bore ablation crater profiles cr eated on bovine corneas using 20 shots at various laser repetition rates..............................................................................................783-15 Scanning ablation crater pr ofiles created on bovine cornea s using 25 shots at various laser repetition rates......................................................................................................... ..793-16 Ablation profiles in PMMA created at 1 Hz for various numbers of laser pulses.............803-17 Ablation depths ( m) in PMMA created at 1 Hz for various numbers of laser pulses.....803-18 Average ablation rates ( m/shot) in PMMA created at 1 Hz for various numbers of laser pulses................................................................................................................... ......813-19 Fluence profile of the Alcon laser beam in mJ/cm2...........................................................813-20 PMMA ablation rate (nm/shot ) verses laser fluence (mJ/cm2) for various numbers of laser pulses................................................................................................................... ......82

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11 3-21 Ablation profiles in PMMA created with 25 laser pulses for vari ous laser repetition rates.......................................................................................................................... ..........823-22 Ablation depths ( m) in PMMA created with 25 laser pulses for various laser repetition rates............................................................................................................... .....833-23 PMMA ablation rate (nm/shot ) verses laser fluence (mJ/cm2) for various laser repetition rates............................................................................................................... .....833-24 PMMA stretched spiral (S) and revers ed spiral (R) ablation profiles at 60 Hz.................843-25 PMMA stretched spiral (S) and reversed spiral (R) ablation profiles at 230 Hz...............843-26 PMMA stretched spiral (S) and reversed spiral (R) ablation profiles at 400 Hz...............853-27 PMMA ablation depths ( m) for the stretched spiral and reverse spiral algorithms at each ablation rate............................................................................................................. ..863-28 PMMA ablation depths ( m) for 60, 230 and 400 Hz for each algorithm........................863-29 PMMA combined ablation profiles for 60, 230 and 400 Hz.............................................873-30 PMMA combined overall ablation depths ( m) for 60, 230 and 400 Hz..........................873-31 PMMA ablation rate (nm/shot ) verses laser fluence (mJ/cm2) for various laser repetition rates............................................................................................................... .....883-32 Representative pre-ablation profile image of a bovine eye...............................................883-33 Representative post-ablation profile image of a bovine eye..............................................893-34 Representative pre-ablation profile image of a bovine eye...............................................893-35 Representative post-ablation profile image of a bovine eye..............................................903-36 Representative stretched spiral and revers e spiral ablation profiles for a paired set of eyes........................................................................................................................... .........903-37 Representative differential ablation profile (stretched spiral minus reverse spiral) of a bovine eye..................................................................................................................... .....913-38 Average differential ablation profile (stretched spiral mi nus reverse spiral) image of a bovine eye created at a lase r repetition rate of 92 Hz.....................................................924-1 Location guide for SEM and TEM microscopy samples...................................................974-2 High magnification H&E-stai ned microscopy images of the ablation crater for laser ablations created at 60 Hz and 400 Hz...............................................................................97

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12 4-3 SEM images of ablation craters created at 60 Hz and 400 Hz...........................................984-4 TEM images of bovine cornea cross-secti ons following laser ab lation at 60 Hz and 400 Hz......................................................................................................................... .......995-1 Schematic of the sub-ablative experi mental setup using 193-nm perturbation...............1145-2 Schematic of the sub-ablative experi mental setup using 355-nm perturbation...............1145-3 Schematic of the sample cell used in the sub-ablative experimental setup.....................1155-4 Representative profile of the 193-nm perturbation beam................................................1155-5 Example of oscilloscope out put for 193-nm perturbation...............................................1165-6 Average extinction coefficient ( N) and standard deviat ions for 0.55 mJ/pulse energy and time step with 193-nm perturbation of collagen solutions............................1205-7 Average extinction coefficient ( N) and standard deviat ions for 0.77 mJ/pulse energy and time step with 193-nm perturbation of collagen solutions............................1215-8 Average extinction coefficient ( N) and standard deviat ions for 0.93 mJ/pulse energy and time step with 193-nm perturbation of collagen solutions............................1215-9 Average extinction coefficient ( N) and standard deviat ions for 1.05 mJ/pulse energy and time step with 193-nm perturbation of collagen solutions............................1225-10 Average extinction coefficient ( N) and standard deviat ions for 0.93 mJ/pulse energy and time step with 193-nm pe rturbation of amino acid solutions........................1225-11 Average number densities (bonds/ml) of peptide bonds and standard deviations for 0.55 mJ/pulse energy and time step with 193nm perturbation of collagen solutions.....1245-12 Average number densities (bonds/ml) of peptide bonds and standard deviations for 0.77 mJ/pulse energy and time step with 193nm perturbation of collagen solutions.....1245-13 Average number densities (bonds/ml) of peptide bonds and standard deviations for 0.93 mJ/pulse energy and time step with 193nm perturbation of collagen solutions.....1255-14 Average number densities (bonds/ml) of peptide bonds and standard deviations for 1.05 mJ/pulse energy and time step with 193nm perturbation of collagen solutions.....1255-15 Number density change per photon for each pulse energy with 193-nm perturbation of collagen solutions........................................................................................................1265-16 Transmission ratio with resp ect to time during laser pulse..............................................1285-17 Example of oscilloscope out put for 355-nm perturbation...............................................128

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13 5-18 Average delay for 355-nm perturbation...........................................................................1295-19 Average extinction coefficient ( N) and standard deviations for 0.48mJ/pulse energy and time step with 355-nm perturbation of collagen solutions........................................1315-20 Average extinction coefficient ( N) and standard deviati ons for 1.2 mJ/pulse energy and time step with 355-nm perturbation of collagen solutions........................................1316-1 Experimental setup for mass spectrometry......................................................................1356-2 Average background spectra ov er entire analyzed range................................................1366-3 Average background spectra over range of activity........................................................1376-4 Average background and ablation spectra.......................................................................1386-5 Average background and ablation spectra focused around 42 amu peak........................1396-6 Simplified collagen structure of the human cornea taken from reference 8....................1406-7 Structure of the 42-amu molecular fragment...................................................................140

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14 LIST OF ABBREVIATIONS amu Atomic Mass Units APD Ablative Photodecomposition ArF Argon Fluoride Laser ECM Extracellular Matrix H&E Hematoxylin and Eosin LASIK: Laser Assisted In-Situ Keratomileusis MMA Methyl Methacrylate PET Poly(Ethylene Terephthalate) PMMA Poly(Methyl Methacrylate) ppb Parts Per Billion PRK: Photo-Refractive Keratectomy SEM Scanning Electron Microscopy TEM Transmission Electron Microscopy TC Tropocollagen UV Ultraviolet

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15 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ArF EXCIMER LASER CORNEAL ABLATION: EFFECTS OF LASER REPETITION RATE AND FUNDAMENTAL LASE R-TISSUE COUPLING By Leia Megan Shanyfelt August 2008 Chair: David Hahn Major: Mechanical Engineering Several topics in excimer laser corneal ablation remain unresolved, ranging from fundamental to practical. The ro les that photothermal and photoch emical processes play in the mechanism of corneal ablation remain a topic of research, including lase r-tissue coupling below the ablation threshold. Goals of the present work are to investigate the mechanism of photoablation and to assess whethe r bovine corneal ablations genera ted at laser repetition rates up to 400 Hz are comparable to ablations perf ormed at current clinical rates (60 Hz). A combination of experiments was implem ented, including ablation plume dynamics, corneal ablation profiles and high-resolution microscopy. Using white-light interferometry analysis, no statistical differen ce was found between corneal abla tion profiles created at 60 Hz and 400 Hz. Using plume imaging and transmi ssion studies, the bulk ablation plume was found to dissipate on a time-scale less th an the pulse-to-pulse separation for repetition rates up to about 400 Hz. A persistent, diffuse component of the ab lation products was observed to be comparable at both rates. Microscopy did not reveal signs of thermal tissue damage for repetition rates up to 400 Hz. Ablations performed on PMMA did not reve al repetition rate effects. Ablation pattern algorithm reversal and plume extr actor addition were analyzed for potential effects on the clinical outcome. Increasing laser repetition rate s for clinical applications appear feasible.

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16 Sub-ablative fluences utilizing 193-nm and 355-nm perturbations yielded insight into photochemistry of collagen and amino acids. Ami no acid solutions were not permanently altered by either wavelength. For collagen solutions, an average of 28 photons at 193 nm was required to break a peptide bond. 355-nm perturbations result ed in an average of 508 photons required to break a peptide bond. A dynamic photobleaching occurs in both amino acid and collagen solutions at both wavelengths and is greater at 193 nm than at 355 nm, resolving by more than tens of nanoseconds but less than tens of sec onds. Permanent changes induced in the collagen samples are due to scission of peptide bond. Mass spectrometry experiments analyzed the ablation products in the ablation plume. The experiments indicate a primarily photochemical ablation mechanism with peptide bonds being the primary chromophore.

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17 CHAPTER 1 BACKGROUND Excimer Laser Refractive Surgery Excimer laser refractive surgery is a techniqu e that is used with a goal of permanently correcting an individuals visi on by reshaping the eyes cornea, thus reducing or eliminating dependence on corrective lenses. Over the past two decades, this type of surgery has grown immensely in popularity. To date, more than 8 million procedures have been performed nationally and more than 17 million have been performed globally. Currently excimer laser refractive surgery is the most popular option for su rgically correcting poor visual refraction [1]. Human Cornea The cornea is the curved structure overlaying th e pupil and iris of the eye. The cornea (1/6 of the outer tunic), together with the sclera (5/6 of the outer t unic), forms the outer fibrous tunic (covering) of the eye, which provides support and pr otection for the eye. The iris is the part of the eye that gives it its color. The iris can tens e or relax to vary the amount of light that is allowed through the pupil, which is the dark central region of the ey e. When the iris relaxes, the pupil dilates, and more light is allowed into the eye. When the iris tenses, less light is allowed into the eye. Behind the iris and pupil is the lens. The cornea focuses light through the pupil and onto the lens of the eye, which in turn focuses light onto the re tina. Together, the cornea and the lens provide the refractive prope rties of the eye, with the ma jority of the refraction being performed by the cornea. The optic nerve transm its the visual information to the brain. The cavity of the eye behind the lens is filled with vi treous humor which retains ocular pressure [2, 3]. Figure 1-1 is a schematic of the general placem ent of the cornea, pupil, iris, lens, sclera, vitreous humor and optic nerve of the human eye [4].

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18 The cornea is a sensitive, tr ansparent (from 400 nm to 1200 nm wavelengths), avascular structure whose anterior (front) surface remains smooth and clean via the lacrimal (tear) glands and blinking action. The corneal geometry is asy mmetric; the average hori zontal diameter is 12.6 mm, while the average vertical diameter is 11.7 mm. Additionally, the po sterior surface is less spherical than the anterior surface, thus the cornea ranges in thickness from approximately 500 microns at the center of the cornea to approximate ly 700 microns at the edge of the cornea. The approximate radius of cu rvature is 8 mm [2, 3]. The cornea itself is made up of fi ve distinct layers. From anteri or (front) to posterior (back) they are the epithelium, Bowmans layer, st roma, Descemets Membrane, and endothelium. These layers are shown in Figure 1-2 [4]. The epithelium and the endothelium are the anterior and posterior membranes, respectively. The epithe lium is a protective cell ular layer that is a barrier between the cornea and the external envi ronment. Bowmans layer consists of densely packed collagen fibers arranged parallel to the corneal surface. This la yer is thought to provide additional structural integrity to the cornea. The stroma, which makes up 90% of the corneal thickness, also consists of collagen fibers, though less densely packed than in Bowmans layer. Descemets membrane and the endothelium provide posterior protection for the eye. The endothelium prevents fluid from within the eye from enteri ng the cornea [2, 3]. As explained above, the main target for cornea l tissue removal in photor efractive surgery is the stromal layer. The corneal stroma is made up of approximately 80% of water and about 20% collagen. The collagen and water make up a co mplex composite material, the extracellular matrix (ECM), which gives the cornea its structur al integrity [5]. The collagen matrix structure consists of regularly-spaced collagen fibrils wh ich are of approximately equal diameter, arranged in layers of parallel lamellae. The stroma is made up of about 300 lamellae (bundles) of

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19 parallel fibrils, with each bundle of fibrils ar ranged parallel to the ad jacent bundle [2, 3, 6]. The reported values of the diameter of the fibrils ra nges considerably, namely from 20 to 34 nm. The center-to-center spacing of the fibrils is 64 nm [5]. In the central region of the cornea, the diam eter is considered independent of corneal hydration [2, 3, 6, 7]. The charac teristic thicknesses of the lamellae are approximately 1 m [5]. The detailed composition and morphology of the corneal tissue dictate the optical properties of the tissue, which in turn establish the internal volumetric distribution of energy of incident laser pulses. Collagen is a right-handed triple helix structure, as shown in Figure 1-3, obtained from [5]. Each strand of the helix is made up of amino acids bonded in series by peptide (CN) bonds into a left-handed helix. The three strands are bonded togeth er by covalent crosslinks into the tropocolla gen (TC) molecule. The TC molecu les are 1.51 nm in diameter and 290 nm long. The TC molecules organize to form the microfibril, which is approximately 3.5 nm in diameter and made up of 6 TC molecules bound by covalent cross-links. The microfibrils then organize to form the collagen fibrils of the ECM [5, 8]. For corneal collagen, each stra nd of the helix is made up of an amino acid chain which generally repeats itself. The amino acid glycine (C2H5NO2) appears about every third amino acid, as shown in Figure 1-3. Gene rally, the amino acids proline (C5H9NO2) and hydroxyproline (C5H9NO3) occur every third amino acid as well; however, trace acids may appear in their stead. Generally, corneal collagen (C12H17N3O4) can be considered to be a repeating structure of glycine-proline-hydroxyproline. A single repeating unit is modeled in Figure 1-4 [5, 8]. When ablated, the immediate underlying st roma becomes disrupted and may become thermally damaged. Notably, at temp eratures above approximately 60oC, coagulation and

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20 denaturing of collagen can occur. Denaturing of collagen occurs when the temperature rise increases the kinetic energy of the collagen molecules such that weak hydrogen bonds and Vander Waals interactions are overc ome. This results in destabili zation of the right-handed helix of the TC molecule. These effects can be observed using TEM microscopy. The denatured collagen fibril is longitudinally s horter and has thicker cross-sectio ns. Disruptions in the collagen structure occur, and a pulling t ogether of fibrils can be seen. This causes the inter-fibriller distance at the ablation surface to become shorter, and thus the density of collagen fibrils would be expected to increase [3, 5, 9]. The level of disorganization and fiber densification observed with high-resolution microscopy may therefore be considered a measure of thermal damage to the underlying tissue [9]. Thermal denaturation of collagen depends on both the tissue temperature and time of exposure. For short thermal exposure times (nsms range), it has been found that the temperature to denature collagen is in excess of 100oC [5]. Venugopalan and co-workers attempted to model the effect of various laser para meters, including pulse duration a nd laser irradiance to determine the zone of thermal energy as a function of th e Pclet number [10]. Un fortunately, a lack of knowledge of the ablation mechanism limited the model, as acknowledged by the authors. A model of thermal damage induced by 193-nm i rradiation that takes into account current understandings, including dynamic optical properties and Beer-Lambert law deviation, still remains unavailable. Thermal damage should be considered in the context of higher laser repetition rates, as less time is available be tween laser shots for en ergy to dissipate via conduction within the corneal stroma. Thus en ergy may accumulate for each laser pulse, and result in higher nearby tissue temperatures, wh ich may result in grea ter underlying tissue damage.

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21 Clinical Implementation Since the first procedure in 1988, refractive vi sion surgery using laser ablation (removal) of tissue has become very popular [11]. Approxi mately 45% of Americans have poor refraction, with 25% of those being myopic (nearsighted), 20% being hyper opic (farsighted) and 35% being astigmatic [12]. Photorefractiv e keratectomy (PRK) and laser in situ keratomileusis (LASIK) are methods of visual correction known as refractiv e surgery. Both PRK and LASIK are forms of keratomileusis, a technique that uses a laser to reshape the cornea about the optical axis to obtain the correct corneal geometry, thereby changing the corneal refractive properties to achieve optimal vision for the patient [3]. Myopic individual s have corneas that are too steep, resulting in a visual focal point in front of the retina. In order to correct for this, the lase r is used to flatten the corneal surface. Conversely, Hyperopic individuals have corneas that are too flat, resulting in a visual focal point behind the re tina. Hyperopia is corrected by using the laser to steepen the cornea [4]. Though the principles of PRK and LASIK surger ies are generally the same, the two differ in their application. In PRK application, the epithelium over the ablation area is removed chemically, mechanically (scraped or brushed away) or by the laser itself, and the exposed corneal tissue is ablated. Both Bowmans layer and the stroma are ablated in th is operation. In LASIK, suction is applied to the anterior surface of the cornea, and a microkeratome is used to cut an anterior corneal flap that includes both the epithelium a nd Bowmans layer. The flap is moved aside, and the underlying st romal tissue is ablated, and the corneal flap is then replaced [12]. It is noted that while the epithelium regenerates quickly, Bowmans layer does not regenerate at all following PRK surgery. This di fference results in an advantage of LASIK over PRK, since there is less scarring and regression when Bowmans layer is preserved [11]. One study of six myopic patients dete rmined that the absence of Bo wmans layer after PRK does not

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22 hinder re-epithilialization, which is typically comp leted during the first post -operative week [13]. Some advantages of LASIK compared to PRK include less scarring, quicker results, less pain, and less post-operative medication [4]. A wide range of reported experimental ablation rate data has been summarized and plotted (Figure 1-5) by Fisher et al. and indicates laser fluences ranging from about 25 to 700 mJ/cm2 with corresponding ablation rate s from threshold to about 1.4 m/pulse [14]. A review of current FDA-approved LASIK systems reveals a wi de variety of average laser fluences on the current market, ranging from about 100 to 250 mJ/cm2, although the peak fluence with Gaussian beams may be in excess of 500 mJ/cm2 [24]. As demand for the procedure has increased, th e surgical procedures and devices have evolved, and laser photorefractive surgery continues to be studied for ways to improve clinical outcome and convenience. Accordingly, accuracy and precision, as well as patient comfort, must be considered in these endeavors. Clinical Issues While much work has been done to improve ex cimer refractive surgery over the last two decades, many issues are as yet unresolved: The surgical process is a closed-loop syst em. The number of shots to achieve a specified correction is pr ogrammed into the computer prior to surgery, and no feedback loop exists to correct for actual conditions. The number of shots delivered by the excimer laser is based on an aver age amount of tissue removed for all people, including all races, both sexes and all ages. Of course, everyones physiology is unique, so using such a general parameter is not idea l. Introduction of system that uses actual conditions, partic ularly corneal hydration, to adjust the prescribed number of shots would be useful Hydration is known to affect the etch depth per incident laser pulse Specifically, cornea dehydrat ion leads to an increase in laser ablation rate [25]. Fisher a nd co-workers used a confocal Raman spectroscopy apparatus to assess corn eal hydration in bovine eyes, noting significant changes in clinically-relev ant time scales of minutes [28].

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23 The mechanism of corneal tissue remova l is not fully understood. A detailed knowledge of the photoablation of corneal ma terial could lead to improvements in surgical lasers, algorithms and post-operativ e care. This topic is discussed in detail in a later section. Using faster laser repetition rates to re duce overall procedure time is an obvious advantage from a patient perspective. Much of the present research is devoted to this topic, as one cannot simply incr ease the laser repetition rate without investigating the prospec tive consequences. These include potential changes in the ablation rate and increases in the damage to underly ing and surrounding tissue due to a possible increase in underlying tissue temperature. The algorithms used to genera te the laser patterns used to remove corneal tissue are not yet perfected. The basic model used to develop ablation algorithms was the blow-off model, which is based on the Bee r-Lambert law. Inherent in the blow-off model are the assumptions that Beer-Lambert applies, that there is a finite threshold fluence, and that material removal begins after the laser pulse is concluded. The second two assumptions are known to be va lid for the case of corneal ablation by an ArF laser. The blow-off model pr edicts the following etch depth: th o aln 1 (1-1) In the above equation, is the etch depth, a is the absorption coefficient, O is the incident fluence, and th is the threshold fluence [5]. A group in Spain has devoted much effort to identifying and resolv ing potential influences on the guiding algorithms, including Beer-Lambert law app licability, beam prof ile considerations, non-normal beam incidence, reflection losses and laser polarization. It was discovered that ablation does not follow B eer-Lamberts law, which is fundamental in the blow-off model, and that correcti ons should be implemented in surgical algorithms to correct for this deviati on. Additionally, loses in energy due to reflection and non-normal incidence were found to reduce the predicted ablation depth [29]. Recently, Fisher & Hahn developed a more current model that takes into account a dynamic tissue absorption coefficient and pr edicts the variation in etch depth with corneal hydration [14] Improving algorithms may result in better outcomes than the current national norm of approximately 90% of patients achieving 20/40 vision or be tter and approximately 65% achieving 20/20 vision or better [34]. The healing process after the surgery c ould also be improved. Eiferman and coworkers noted in an early study that ther e are no adverse eff ects on the eye caused by the laser itself. It is noted that the high absorption coefficient of the cornea for 193-nm radiation limits the penetration of the incident pulse to within a cellular layer of the ablated tissue. They also de termined that endothelial cell counts an intraocular pressure remained unchang ed from pre-surgery conditions and important observation because endothelial cells do not regenerate like their

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24 epithelial counterparts. Also, contrast sensitivity and brightness acuity were not degraded at 3and 6months post-operat ive [13]. Corneal haze, one of the more common side effects of laser ablation, may appear after PRK or LASIK, and usually dissipates during the first 12 m onths post-operative. Other complications include glare, halo and decreased contrast Healing is generall y faster after LASIK than PRK, and development of corneal ha ze is less common [12]. Much research has been devoted to the topi c of corneal haze, which occu rs when keratocytes in the cornea become fibroblastic and lose their transparency and act as light scattering centers within the cornea. Haze is a comm on post-operative occurance in the first four months following surgery, and typi cally diminishes over time [11, 35]. In many cases the first refractive surger y results in under-correction, and a second surgery is needed to complete the refrac tion [12]. The larger the refractive change required, the greater the chan ce that a secondary surgery will be required. This fact couples into the closed-loop system discus sion above. Any errors in the algorithm and number of shots prescribed will grow with the length of the surgery. Also, hyperopic individuals are more likely to require enhancement than myopic individuals. As it can take se veral months for ones vision to settle into an outcome; hence, enhancement surgeries are typical ly not performed for 3 months. Ideally, secondary surgeries to refine the results would be unnecessary in all cases [34, 38]. Polymer Ablation It is also useful to study the larger body of polymer ablation to gain additional insight into tissue ablation. In 1983, Srinivasan at the IBM Research Center pioneered the technique he coined ablative photodecomposition, or APD, wh ich eventually led to photorefractive surgery as we know it. This new etching technology, whic h was made possible by the advent of the UV excimer laser in the late 1970s, us es ultraviolet (UV) radiation to sculpt the targeted material with great precision. Excimer lase rs have diatomic species Ar2 *, Kr2 *, Xe2 *, ArF, ArO, KrF, KrCl, XeCl and XeF, for example as the activ e medium inside the laser cavity. The term excimer laser comes from excited dimer. Techni cally, excited dimers are homonuclear, such as Ar2 *. Many excimer lasers are actually excited excipl ex lasers which are heteronuclear, such as ArF; however, the term excimer is used for both dimer and exciplex varieties. These gas lasers are ultraviolet pulsed lasers with pulses on the order of nanosec onds with powers ranging from 1 to 100 W [39, 40].

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25 APD was and is still attractive due to its high spatial resolution and lack of thermal damage to surrounding material. The early work on th e topic was performed on organic polymers, particularly poly(ethylene tere phthalate), or PET; polyimide trademark name Kapton; and poly(methyl methacrylate), or PM MA. It was first noted how clean ly far-UV laser light etched the surfaces of these organic polymers, and early investigations focused on determining the etch rate (material depth removed per shot) of thes e materials. From these beginning experiments, interest in the topic boomed and deepened, w ith identified potential applications in microfabrication, microsurge ry and art restoration. The question of exactly how the photodecom position occurs has since been debated. Experimentalists have examined the effects of UV ablation with a wide variety of parameters such as environment, laser wavelength, laser pulse width and laser pu lse energy on the specific ablation mechanisms of PET, Kapton, PMMA and others. Additionally, many different types of excimer lasers have been used in these studies, including KrF (248 nm), KrCl (222 nm), ArF (193 nm) and XeCl (308 nm). With the variety in UV radiation source comes a spread of laser fluence, excitation wavelength and pulse dura tion. This is an extremely large array of experimental conditions. The first proposed mechanism for APD with 193-nm light on PMMA was a photochemical one which predicted that after reaching a threshold energy (fluen ce) required for smooth etching, the laser pulse energy causes bonds to break w ithin the polymer strands thus increasing the specific volume of the material The excess energy remaining fr om the photon after the bonds are broken vibrationally, rota tionally and translationally excites the polymer fragments, which are ejected from the polymer surface [39]. Additional investigators contended that the mechanism is entirely thermal, and not photochemical at all [ 41]. Still more presented mechanisms are reported

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26 to involve both thermal and photochemical in teractions [42]. These disagreements among early authors were indicative of a prolonged di fficulty in specifying the exact mechanism of APD. Over the next two decades the argument has co ntinued with no resolution. The mechanism of ablation of polymers by UV radiation is st ill unknown. Many groups, including chemists and physicists, across the globe have studied the topic using a vast array of techniques including laser-induced fluorescence, mass spectrometry, emission spectra and Raman spectroscopy. Still no agreement on the mechanism of ab lation has been reached [45, 46]. Current theories include photochemical and photothermal mechanisms. In the case of photochemical theories, photon exc itation directly results in bond breaking. Both single and multi-photon absorption models have been proposed. In the case of photothermal theories, photon excitations lead directly to thermal br eakdown of the polymer bonds. Many other theories incorporate combinations of these two [45, 46]. Part of the difficulty is the vast array of parameters that alter the experiments. It is generally agreed that changes in excitation wavelength, pulse duration, laser fluence, and th e properties of the specific polymer studied, including refractive index and mol ecular weight all influence the precise mechanism of ablation. In the experiments presented in the current study, a 193-nm ArF laser was used to etch PMMA. PMMA is a polymer crea ted of monomer units of MMA. The arrangement of a PMMA molecule is depicted with two of the re peating monomers in Figure 1-6 below [47]. PMMA has successfully been used in the past as a surrogate model for laser refractive surgery. Past studies have employed the polymer to predict the impact of surgery on corneal shape and to estimate post-operative corn eal surface roughness. Consider the algorithm limitations outlined by Jimenez and co-workers as described above. Variations based on things

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27 such as reflection loss and non-normal incidence are expected to have the similar impact on PMMA as on a human cornea [26, 48]. It is important to note the differences betw een the ablation characteristics of the two materials, namely polymers and corneal tissue. Fi rst is the ablation threshold. Ablation in the human cornea begins between 40 and 60 mJ/cm2, while ablation in PMMA begins around between 35 and 80 mJ/cm2 for ns-pulsed ArF excimer lasers Additionally, the ablation rates (amount of material removed per laser pulse) are unequal. One study found the ratio to be 1.8 microns of corneal material removed per 1.0 micron of PMMA removed by comparing overall ablation depths generated on PMMA by a flying s pot laser system with the published corneal overall ablation depths provided by the vendor. It is important to keep these factors in mind when comparing ablation patterns created on th e different materials; accordingly, one would expect a corneal ablation profile to be slightly wider and much deeper than a profile created on PMMA using the same algorithm [48, 49]. For a co mparable laser system as that used in the current experiments, the ablation rate of PMMA was measured to be 0.47 m/pulse [50]. The ablation of PMMA is non-lin ear as a function of fluence. The semi-log plot of depth verses fluence has a lazy S shape with three dis tinct zones. In the first zone at lower fluences, the etch rate increases slowly with fluence, as the threshold value for ablation is reached. The second zone is a linear region wher e etch rate increases consisten tly with increased fluence. At high fluence, this linear regime ceases, and the etch rate begins to level off, or even decrease. An example plot of this behavior obtained from Sr inivasan and Braren is presented in Figure 1-7 [49].

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28 Corneal Ablation Very soon after APD was described for polymer s, ablation of tissue by UV lasers began to be investigated for possible applications, in cluding corneal refrac tion correction. A thorough understanding of the physics and mechanisms of co rneal ablation and the pot ential role of laser repetition rate remains a topic of research [5, 28, 31, 51]. As the exact mechanisms of photoablation remain unidentified, it is critical to verify that in creasing the laser repetition rate does not reveal any differences in surgical outcome or underlying corneal pathology. The lasertissue interaction may be generalized as a dyna mic process during which the corneal optical properties are perturbed by the la ser beam during the time-course of the ablating laser pulse. In the context of a photochemical ablation proces s, photocleavage of collagen bonds leads to subsurface expansion of the corneal tissue matrix and subsequent stress that drives the ablated tissue from the surface in the form of the well-known ablation plume [14, 55]. As previously described, the human cornea is comprised of mainly collagen and water. For corneal ablation, a main parameter is the tissu e absorption coefficient. The tissue absorption coefficient dictates how strongly the incident laser energy is ab sorbed into the tissue and how deeply the energy will penetrate. In corneal ablation, where light scattering by the transparent tissue is negligible, the optical penetration dept h of the incident radiat ion is defined as the reciprocal of the absorption coefficient. The optic al penetration depth is the depth to which the laser energy will travel, and which the tissue is affected. In order to understand ablation of corneal tissue, the abso rption coefficient for both collagen and water must be understood. At room temperatures, absorption by water of 193 nm is negligible as compared to that of collagen. The absorption of collagen at 193 nm is signi ficant, with an absorption coefficient of approximately 2 x 104 cm-1. The most important chromophore at 193 nm is the peptide bond which separates the constituent amino acids. The peptide bond absorption peaks roughly at 190

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29 nm. The absorption of laser light by collagen is highly sensitive to wavelength, as the absorption of collagen-based tissues drops at least 100 ti mes when wavelengths of 240 nm are used as compared to 193 nm [5, 51]. The energy supplied by the ablation laser cause s thermal and mechanical changes in the corneal tissue, which result in significant changes to the tissue optical properties. In fact, the absorption coefficients of both water and collagen are enhanced by addition of the laser energy to the tissue. The absorption coefficient of wa ter is enhanced as the absorption band (peak) normally located at 160 nm shifts to longer wave lengths, resulting in a non-negligible absorption coefficient of water during the ablation proce ss. For a volumetric energy density of 2 kJ/cm3, the absorption coefficient may reach 104 cm-1 [60]. The absorption of collagen was found to be increased by exposure to laser en ergy at 193 nm for times up to 10-4 seconds after the laser pulse [61, 62]. Pettit and Ediger found that the absorption coefficient at 193 nm of stromal tissue to be 40,000 +/10,000 cm-1, a value 10 times higher than previously reported values [63]. Fisher and Hahn found the value to be 16,000 cm-1, Yablon et al found the value to be 19,000 cm-1 and Munnerlyn et al found the value to be 37,800 cm-1, all confirming the work of Pettit and Ediger, namely that the absorption coefficient is signi ficant and maby be enhanced during the ablation process [8, 64, 65]. The ArF excimer laser ablation of corneal tissu e is considered to en tail considerable bond breaking and the ejection of molecular fragments. The dissociation energies (eV) of selected molecular bonds in tissue are identified in Table 1-1. These en ergies may be compared to the 6.4 eV photon energy (E = h ) of the 193-nm ArF excimer laser us ed in photorefractive surgery. The energy of a single photon of ArF irradiation is sufficient to break all of the molecular bonds of Table 1-1, with the exception of C=O, as adapted from Vogel & Venugopalan, 2003. The

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30 photon energy of the ArF excimer lase r is also larger than that of other UV lasers, such as the KrF excimer (5.0 eV) and the XeCl excimer (4.0 eV). In order for bond breakage by the excimer laser to be the primary means of photodecomp osition, the rate of bond breakage by the UV laser must exceed the rate of recombination by the broken bonds in the decomposed species [5]. The decomposition of collagen into fragments re sults in a larger volume of material, which in turn generates a local stress within the cornea. This internal stress then results in the ejection of the fragmented corneal material. As previ ously indicated, the pep tide bonds that link the constituent amino acids of the collagen are the primary chromophor es, and is thus the primary candidate for the specific corneal bonds that are broken due to exposure to the excimer laser energy. It is thought that this is an important piece of the mechan ism of tissue removal in corneal photorefractive surgery; however, this does not form the complete picture. It is also generally thought that photothermal (or ther mal) effects play a role in the ablation mechanism. Energy exceeding the bond energies increases the kinetic energy of the tissue, resulting in temperature increases of the dissociated products A portion of this energy may also transfer to the water in the cornea, increasing its temperature and thus allowing it to become a greater chromophore as its absorption coefficient rises. Vaporization of water may then occur [5]. Others found that the corneal surface temperature exceeded 100oC when ablated with an ArF with a fluence of 80 mJ/cm2, further indication of significant thermal c ontributions [66]. Howeve r, due to the brief nature of the laser pulse (tens of nanoseconds ), thermal diffusion to surrounding tissues is minimized. This coupled with the short penetrati on depth lead to minimal thermal damage of the underlying stroma [5]. Another important feature of corneal ablation is the ablation threshold. At low energies, by definition the laser exposure does not ablate the cornea. At a specific radiant exposure

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31 (approximately 30 mJ/cm2), known as the ablation thresh old, tissue removal begins. At higher energies, the etch depth of the ablation increases with radiant exposure [5, 17, 48]. For a comparable laser system as that used in this experiment, the ablation rate of bovine corneas was measured to be 0.90 m/pulse [50]. Additionally, Fisher and Hahn reported values of the absorption cross section of peptide bonds and amino acids in collagen solutions. The overall absorption coefficient for collagen was found to be 1.19E-17 cm2, while the contribution by peptid e bonds was calculated as 1.14E-17 cm2 and that by amino acids was calculated to be 4.74E-19 cm2 [8]. Another parameter of interest is the quantum yield. The quantum yields and relative concentrations of collagen components can be used to determine a probabil ity of photochemical damage. This probability has been reported as 300 cm-1M-1 for peptide bonds, 4.7 cm-1M-1 for glycine and 2.5 cm-1M1 for proline. These numbers were generated on the assumption that glycine made up 33% of the collagen and proline made up 13% of the collagen. The probability for photochemical damage is much greater for peptide bonds than for amino aci ds. The same study also determined that 60% of peptide bonds within corneal collagen may be de stroyed with each incident laser pulse. This, along with the probabilities, supports the s upposition of a photochemical mechanism [51]. Experimental Methods and Studies Plume Dynamics With each incident excimer laser pulse, the abla ted corneal material is ejected from the eye surface to form the ablation plume. It is essent ial to examine the dynamics of this plume to determine if the physical removal of tissue during ablation is altere d as the laser pulse rate is increased to values higher than current clinical rates. Excess material li ngering over the surgical plane may alter the ablation process, notably by attenuating (shielding) the laser pulse energy and thus decreasing the amount of corneal tissue removed. Also, the excess material could settle

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32 back down onto the corneal surf ace, also resulting in underablation of the cornea. This phenomena is generally associated with wide beam s [5, 65, 67]. Accordingl y, it is desirable that the ablation rate provide adequate time for th e ablation plume from one shot to sufficiently dissipate before the next laser shot occurs. The ablation plume is made up of the materi al ejected from the ablation site, and may include water vapor, water droplets, gaseous or ganic products and particulate tissue fragments. Hahn and co-workers found water droplets to be the dominant constituent present in corneal ablation plumes [68]. There is a delay between the incident laser pulse and the beginning of the ejection of the plume, which has been measured as 70 ns for ArF ablation of corneal tissue. The velocity at which the plume ejects is over 600 m/s for fluences in the range of 300 mJ/cm2 to 1 J/cm2, and exerts a recoil stress upon the cornea. After 500 ns, the plume slows to approximately 350 m/s [5]. The ejected material rises from the entire ablated surface area a nd immediately necks into a typical mushroom cloud. This effect is due to Bernoullis la w that requires that the sum of the static pressure and the dynamic pressure (the product of one-half the density) and the square of the flow velocity be constant. As the velocity of the flow is quite large, the pressure in the flow relative to the surroundings becomes sma ll; thusly, the flow from the corneal surface immediately condenses to a tighter diameter. Th e mushrooming at the top then occurs due to turbulent eddy formation [5]. One way to analyze plume dynamics is by imaging the plume as it is ejected from the corneal surface using a camera [67, 69]. The resulting pictures allow the plume to be visually analyzed, including time-evolution of the plume. A second way to analyze the plume is with

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33 transmission studies [70]. Figure 1-8 shows a t ypical transmission experiment setup. In general terms, the transmission is defined as 0) ( I I (1-2) where I() is the intensity of the laser light afte r passing through the pl ume path length, and I0 is the incident lase r intensity. A 100% transmission measur ement corresponds to an optically transparent sample. Therefore, when no material in the sample is present, the transmission would be equal to unity. As more material is added inside the pathlength, the transmission measurement will decrease. In the case of plume dynamics, the plume itself is the probed sample. White-Light Interferometry An important tool for analysis of ablation cr aters is the white-light interferometer. There are two main types of interferometers: Fa bry-Perot and Michelson. Only a Michelson interferometer was used for the present study. Th e Michelson interferomet er, invented by Albert Abraham Michelson, is a division of amplitude in terferometer, meaning that the incident white light beam is split into two separate beams that interfere with each other [71, 72]. A basic Michelson interferometer is diagramed below in Figure 1-9. The incident beam (source) is split using a pa rtially-reflective plate (beam splitter), such that part of the incident beam passes straight through the plate to ward the fixed mirror, and part of the incident beam is reflected at 90o toward the movable mirror. The compensator is an optical piece of the same material and thickness of the plat e, and is used to ensure that both beams pass through the same material thickness. Without this piece, one beam would pass through the plate material once, while the other would pass through it three times. Both portions of the beam recombine at the plate and reflect onto the de tector. The recombined light has a pattern of interference fringes, because the two beams of light have traveled different distances [71, 72].

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34 The interference is a function of the phase difference of the two beams. In the case of a monochromatic light source, th e out-of-phase difference distance t can be related to the pathlength difference x. The dist ance t is the physical differenc e between the image plane from the stationary mirror image and the image plane of the movable mirror image. The distance x can be varied by moving the movable mirror parallel to itself. Moving this mirror changes the pathlength difference of the two beams by twi ce the distance that the mirror is moved. The relation between t and x is theref ore given by the following equation: 2 2 m t x (1-3) When the value of m is an even number, th e images interfere constructively. Likewise, when m is an odd number, the images interfere de structively. This makes intuitive sense if one think about the wave behavior of light. If the two image waves come in together in-phase, the amplitude of the wave will be doubled (constructive interference). If the imag es come in together exactly out-of-phase ( /2 phase difference), the amplitude of the waves will cancel each other out (destructive in terference) [71, 72]. White-light interferometry is more complex; ho wever, the general concepts are the same as that with a monochromatic source. In the expe riments performed in this study, a Zygo NewView scanning white-light interferometer was used to determine detailed surface geometry of various samples. This specific interferometer scans vertic ally by using a piezoelectric transducer to move the microscope objective, rath er than using the movable mirror discussed in the basic interferometer setup. The traditional movable mirror is stati onary in this device. Realize that this type of movement can be related to movi ng the movable mirror, as the pathlength increase of the side beam will be double that of the increase of the main beam. The NewView reflects light off of the sample to be measured and off of a high quality reference surface. Both of these

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35 reflections are recombined onto a solid-state camera. The NewView System itself converts the white-light interference fringes into useful dist ance variations and creates a three-dimensional profile of the measured sample [73]. White-light interferometry has been successfully used to study ablation crates created by a photor efractive surgery la ser system [50]. Mass Spectrocsopy Mass spectrometry has been us ed to identify ablation plum e products. Two studies have analyzed the products from abla ted human corneas. Unfortunatel y, these studies are dated. The technology has progressed in the last decade, as has the understanding of the ablation process [74, 75]. Kermani and co-workers examined the ablati on plume from ablating human corneas with an ArF (193 nm) excimer laser and a KrF (248 nm ) excimer laser at fluences from 5 mJ/cm2 at 1 Hz. The intent of their study was to use mass spectrometry analysis of the ablation plume to glean information about the difference in corn eal ablation performed at different wavelengths and different energies. There are two distinct limitiations in their experiment. First, they had available old laser technology that resulted in poor-quality beams with uneven energy distribution. Second, the mass-sp ectrometry apparatus involved pulling a vacuum on the eye prior to and during ablation. The au thors noted that the vacuum (10-7 Torr) dehydrated the eye prior to ablation, as during the ev acuation process, the mass spectro meter registered water in the system. This causes two distinct problems. It is now well-known that corneal ablation is affected by corneal hydration, affec ting the ablation rate of tissue. It is has also been discovered that water droplets and vapor are c onstituents of the ablation plum e. Therefore, by dehydrating the tissue prior to ablation, the ablation is altered an d the mass spectrometric analysis is incomplete. With todays knowledge and technology, these limitations can be overcome [25, 68, 75].

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36 Kahle and co-workers examined the ablation plume from ablating human corneas with an ArF (193 nm) excimer laser and a Er:YAG (2.94 m) solid-state laser at a fluences of 180 mJ/cm2 and 2.7 J/cm2. The ArF excimer laser had a beam width of 3.5 mm and a repetition rate of 10 Hz. The second experiment was subject to the same limitations as that of Kermani, et al An air pump was used to pull the samples thro ugh to the mass spectrometer through a moisture absorber. It is unclear from the description of the experiment if the eye was dehydrated due to the pull of the pump. However, the same argumen t related above regarding dehydrated plume samples is applicable [74]. Kermani and co-workers found low-amu (less than 20 amu) components in the ablation products, including H2, C, CH, CH2, CH3 and NH2 and higher-amu components including COH, CH2NH2, CH2OH. In contrast, Kahle and co-workers found C10H23ON, C11H24, C12H26, C14H30, C14H24O3, C20H38, C20H14O3 and C21H24 in the ablation products. Th e results of the two studies are not in agreement [74, 75]. As with other ablation experiments, plum e analysis via mass spectrometry has been performed on polymers. Some examples are provid ed here. Grivas and co-workers used time-offlight mass spectrometry to study the dynamics of polyarylsulfone films, which are used in microelectronics. Tsunekawa and co-workers us ed mass spectrometry to study laser ablation of PMMA and PS at 308 nm to study the mechanism of polymer ablation. Hansen used time-offlight mass spectrometry to study the ablation pr oducts of several poly mers, including PMMA. Thus, the use of mass spectrometry as a tool fo r ablation plume analysis has been proven [76 78].

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37 Objectives of the Present Work There are several topics in the field of excimer laser corneal ablation that remain unresolved. These topics range from the fundament al to the practical. The specific mechanism of corneal ablation is not fully unde rstood to date. For example, the roles that photothermal and photochemical processes play remain a topic of research, including the laser-tissue coupling at and below the ablation threshold fluence. A goal of the present work is to investigate the mechanism of photoablation. Anothe r goal of the present work is to assess whether bovine corneal ablations generated at la ser repetition rates of up to 400 Hz are comparable to ablations performed at rates consistent with current clinical laser systems ( 60 to 100 Hz). Increases in laser repetition rate will reduce surgic al procedure time and are therefor e beneficial. Specific research tasks in this proposal are as follows: Plume Dynamics Ablation Profiles Histology Sub-Ablative Study Mass Spectrometry of Ablation Plume

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38 Figure 1-1. Schematic of the hum an eye, taken from reference 4 Figure 1-2. The five layers of the human cornea, taken from reference 4

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39 Figure 1-3. Schematic of the triple-helix st ructure of collagen, taken from reference 5 Figure 1-4. Simplified collagen structure of the human cornea, taken from reference 8 C C N C C N C C N O O O C C C (Hydroxyproline) (Glycine) Glycine Proline Hydroxyproline OH C C C

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40 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0100200300400500600700800Ablation Rate (microns/shot)Fluence (mJ/cm2) Figure 1-5. Ablation rate (microns /shot) verses Fluence (mJ/cm2) summary of reported values in the literature, taken from reference 14 Figure 1-6. PMMA molecule, adapted from reference 47 C CH2 CH3 CH3 C O O C CH2CH3CH3 CO O

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41 Figure 1-7. Semi-log plot of etch depth vers es fluence in PMMA at 193 nm, taken from reference 49 Table 1-1. Selected molecular bond s in tissue and their dissociati on energies (eV), adapted from reference 5 Molecular bond Dissociation energy (eV) Molecular bond Dissociation energy (eV) 7.5 N H 4.1 6.4 C O 3.6 4.8 C C 3.6 4.3 C N 3.0

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42 Figure 1-8. A typi cal transmission experiment setup Figure 1-9. The setup of a tradit ional Michelson interferometer Source Beam Splitter Compensator Fixed Mirror Detector Laser Probed Sample IO I() Movable Mirror

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43 CHAPTER 2 PLUME DYNAMICS The dynamics of the corneal ablation ejecti on plume are outlined and discussed in this chapter. The experiments presented include bot h plume imaging and transmission measurements which are used together to understand possible e ffects of increasing lase r repetition rate on the corneal tissue ablation process. Experimental Setup and Methods In all experiments involving bovine eyes, the specimens were obtained with IUCAC approval (#B220) from the Univer sity of Florida Animal Scien ces Department slaughterhouse. Whole eye globes were extracted immediately following sacrifice (le ss than 20 minutes) and placed in sealed, buffered saline solution-filled pl astic bags and kept at room temperature. Experiments were always performed within severa l hours following animal sacrifice. Prior to all ablations, the corneal surface was mechanically de-epithelialized us ing a scalpel edge. In all studies, the TUI (Santa Clara, CA) Ar F excimer laser output energy was nominally 3 mJ per pulse at the corneal eye plane and th e focal spot size was approximately 0.83 mm. The laser is capable of producing lase r repletion rates from 1 Hz to above 400 Hz. Alcon proprietary software controlled the delivery of a specified refractive correction over a 6-mm treatment zone using a flying spot spiraling algorithm. As in clinical settings, the laser beam was oriented downward onto the bovine eye. The surgical 193-nm ArF excimer laser beam (Laser 1 in Figures 2-1 and 2-2) passes through a beam homogenizer, a focusing lens and a pinhole before entering the scan cube. The scan cube cont ains two orthogonal mirrors that move in concert to generate a correction profile at the corneal plane. After exiting the scan c ube, the beam then passes through a second focusing lens and two mirrors and is projected downward onto the corneal surface. The

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44 bovine eye globe was held in a specially designed holder to keep it static and taut during the procedure. Plume Imaging To assess the ablation plume dynamics, planar laser light scattering measurements were performed. An Andor iStar iCCD camera (1024 X 1024 pixels) was used to record images of the plume. For imaging, a Nd:YAG pulsed solid-state laser (Laser 2) at 532 nm was used to illuminate the ablation plume, as shown in Figure 2-1. A cylindrical lens was used to create a vertical sheet of light whic h passed above the corneal su rface normal to the camera. Using digital delay generators, the pulsed Nd :Yag light was synchronized to the ablating excimer laser pulse. Due to internal delays in the system, the minimum delay between Laser 1 and Laser 2 shots was 121.3 s. The jitter in the system was a pproximately +/50 ns, an order of magnitude lower than the timescales examined in these experiments. By adjusting the delay between the two lasers, the temporal evolution of the ablation plume was imaged for delays ranging from approximately 121 s to 40 ms following the excimer laser pulse, noting that a delay time of zero corresponds to coincident lase r pulses. The method of us ing scattered light to image an ablation plume is well documented and da tes back to the pioneering work of Puliafito and co-workers [67, 69, 70]. Plume Transmission To further analyze the ablation plume evolut ion, transmission measurements through the plume were analyzed. For this experiment, a secondary 193-nm ArF laser beam was used to probe the ablation plume, includi ng interactions with solid phase (i.e. particulate) and gas phase components.

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45 The path of the surgical ArF laser (Laser 1) fo r these studies was identical to that described above. The GAM (Orlando, Florida) excimer laser beam (Laser 3) was split into two paths, considered the incident and transmitted, using a gl ass quartz flat as a beam splitter. The incident beam path passes through the quart z flat, then through a set of neut ral density filters to ensure signal linearity, an aperture to minimize stray light, a 193-nm line filter to eliminate any laserinduced fluorescence signal, and finally into the detector (200-ps rise-time photodiode). The transmitted beam path follows a similar path of optics but is passed through the center of the ablation plume at a height of 2 mm above the corneal surface. Figure 2-2 demonstrates the configuration used for this set of experiment s. This general method has been successfully implemented in the analysis of ablation plume dynamics [70]. The incident probe signal was compared to the transmitted signal to determine the transmission. The ratio of the integrated transm itted signal to the integrated incident signal normalized to the ratio with no plume present de fines the transmission. In general terms, the transmission is defined as 0) ( I I (2-1) where I() is the intensity of the laser light afte r passing through the pl ume path length, and I0 is the incident laser intensity. Thus, the normalized ratio (i.e. plume/no plume) is the transmission through the ablation plume. Using di gital delay generators, the transmission of the plume was determined as a function of time with respect to Laser 1, as in the plume dynamics study above, for delays ranging from 0 s to 40 ms. The transmission of light through the ejected plume couples with the plume images to assess whether the material removal mechanism is altered by residual plume material from previous laser shots. Recalling that the probe laser passes at a distance of 2 mm above the

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46 ablation plane, the period of minimum transmissi on should correlate to the brightest spot in the scattering image (temporally) at this location. Likewise, as the transmission recovers towards unity, the plume should be dissipa ting into the environment. Plume Dynamics Results Plume Imaging Two full sets of images following the temporal evolution of the plume were recorded with the imaging laser (Laser 2) running at 2 Hz. Each image set was performed on a fresh bovine eye. Both sets begin at approximately 120 s and end at over 30 ms with respect to the ablating laser. This recorded temporal evolution of the ablation plume s hows the behavior of the plume ejection and time scale of plume dissipation. This e volution is pictured in Figure 2-3. Each image is a single capture from the iCCD camera. The time scale represents the delay of the image with respect to the surgical laser (Laser 1) pulse From these images, one can see the mushroom cloud of the plume initiate and th en dissolve into the surrounding environment. As observed in the figure, a column of ablated material ejects above the surface, and side vortices develop and expand with time. The column grows, and the vorti ces rise and spread as the plume travels away from the ablation surface, due to the ejection velocity. An important question concerns the potentia l plume effect resulting from increasing the laser repetition rate from 60 to 400 Hz. To answer the question of shot-to-s hot plume effects, the evolution of the plume at 1/60 (16,7 ms) a nd 1/400 seconds (2.5 ms) are compared. Plume images at long times (2.5 ms and 9 ms) are shown in Figure 2-4. At these long time frames, it is difficult to glean an obvious difference between the plumes (particularly in the central region, which corresponds to the surgical beam path). To further analyze the plumes in a quantita tive manner, the average scattering intensity was calculated at each time frame. The average scattering intensity was determined by dividing

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47 the sum of the intensity at all pixels by the to tal number of pixels, th en subtracting off the average intensity of the background (i.e. image with no plume present). The results of these calculations for both sets of da ta are plotted in Figure 2-5. The average scattering intensity decays rapidl y in the first 5 ms after the plume ejection, then begins to level off into a slow decay. This plot correlates well with the images themselves, which show a similar trend. The data indicate a threshold of about 2 ms, beyond which one would anticipate negligible interaction of the in cident laser beam with the ablation plume from the previous pulse. At 400 Hz la ser repetition rate, the pulse-topulse spacing is 2.5 ms, which is consistent for this time scale, he nce no significant effects are expe cted from interactions of the laser pulse with the bulk ab lation plume for laser repeti tion rates up to about 400 Hz. Plume Transmission To further corroborate the imaging studies, tr ansmission measurements were performed to ensure that the laser energy of the ablation be am is not truncated by excess plume material lingering in the beam path. Transmission experi ments were repeated on six bovine eyes. The delay range of the experiment began at 171.3 s up to 1 ms. Three transmission measurements were taken at each delay time per eye. The aver age transmission of the 193-nm probe laser is plotted in Figure 2-6 as a functi on of delay time following the ab lating laser pulse (time=zero), hence the time axis represents th e delay of the probe excimer beam (Laser 3) with respect to the ablation pulse (Laser 1). The error bars on th e plot represent one standard deviation. The transmission profile shows a minimum of about 91% at a delay time between 120 250 s, which corresponds to the passage of the bulk ablation debris thro ugh the probe beam at the probe beam height of 2 mm above the corn eal surface, as shown in the plume imaging study. After the bulk of the plume evolves away, the tr ansmission steadily climbs to 98.4%, rather than

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48 100%. Examination of Figure 2-6 re veals that the transmission valu e of about 98.4% is persistent at the earliest delay times as well, namely between 1 and 10 s. It is noted that these early times are well before the arrival of the bulk ablation plume. In order to examine the cause of this overall decrease in transmission, the ArF probe beam experiments were preformed again, but without a target at the ab lation laser focal plane. This experiment was designed to determine if environm ental factors, namely a build-up of ozone in the ablation area, are the cause of the observe d transmission loss. These control transmission measurements were determined in the same way as their corneal counterpart s, but with no target present. A shutter was placed between the Alcon lase r (Laser 1) and the target area. The ratio of probe laser transmission was calculated by normaliz ing the transmission with the Laser 1 shutter open to the transmission with the Laser 1 shutter closed by alternatively opening and shutting the shutter. Just as with the corneal target, these experiments were performed at various delays. As the probe laser passes through the Alcon laser pa th at different delay times, any effect of enhanced absorption (e.g. Ozone formation) re sponsible for the ~2% transmission drop noted above should be apparent. If ozone formation is responsible for the transmission loss, a maximum transmission of approximately 98% shoul d be achieved. The results in Figure 2-7 do not show such a drop in transmission, indicating that ozone is not the source for the transmission loss. Therefore, additional measurements were pursued. A final set of experiments was performed to fu rther assess the transmission loss of the ArF probe laser through the ablation plume under abla tive conditions. Because the 2% drop in transmission appears to be long lived, one goal was to explore the possi ble effects of ablation rate; hence measurements were recorded at 60 Hz and 400 Hz. As the ability to synchronize the ablating laser and probe laser was not possible at 400 Hz, the transmission measurements were

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49 done without any laser-laser synchronization. The ablating laser was run at either 60 Hz or 400 Hz, and the probe laser was run at a constant rate of 50 Hz. This setup does not have the capability to study the temporal evolution of the transmission, but rather yields an average transmission. This average transmission of 20 pr obe laser pulses was repeated for six ablation sites for each bovine eye samples. These measurements were performed in quiescent air and then repeated with addition of a nitrogen purge cross flow jet. The cross flow was achieved by placing a piece of rigid tube 1 cm from the ablating laser pulse, aligned at the heig ht of the probe laser, as shown in Figure 2-8. A flow of dry nitrogen (10 liters per minute) formed the cross-flow jet. The tube was orthogonal to the direction of the probe laser, with both th e tube and probe laser in the same plane, approximately 2 mm above the target surface. With this configuration, the transmission measurements were repeated. At each repetition rate and flow condition, 2 bovine eye samples were used. It was observed that by adding a cross flow of dry nitrogen to purge the beam path allowed the transmission to reach unity at a time scal e of 1.25 ms. Transmission measurements were recorded for both laser repetition rates (60 and 400 Hz) with cross flow added. The results are shown in Figure 2-9, for both 60 and 400 Hz. With the nitrogen purge flow, the transmission is increased to 100.4% ( =0.49%) and 99.9% ( =0.95%) for the 60and 400-Hz experiments, respectively. Together, the data suggest that a diffuse, gas-phase component accumulates above the ablation surface and slightly attenuates the incoming laser pulse. However, no significant laser repetition rate effect is observed for this diffuse plume co mponent. Clinically, this diffuse component is most likely diminished by the exha ust systems (i.e. plume extractors) that are

PAGE 50

50 present on commercial refractive systems, although the degree to which it might exist is difficult to predict. The average probe transmission value of th e 60 Hz ablation with no flow is again statistically indistinguishable from that at 400 Hz ablation with no flow. Plume Dynamics Summary Qualitatively, the bulk of the ablation plume is observed to dissipate on a time-scale of about 3 ms to about 98% transmission. Likewise, the average scattering intensity decays rapidly in the first few ms after the plume ejection, then begins to level off into a slow decay. At 400 Hz laser repetition rate, the pulse-topulse spacing is 2.5 ms, which is consistent for this time scale, hence no significant effects are expected from interactions of the laser pulse with the bulk ablation plume for laser repetition rates up to 400 Hz. The transmission profile shows a minimum of about 91% at a delay time between 120 250 s, which corresponds to the passage of the bulk ablation debris thro ugh the probe beam at the probe beam height of 2 mm above the corn eal surface, as shown in the plume imaging study. After the bulk of the plume evolves away, the tr ansmission steadily climbs to 98.4%, rather than unity. With the addition of nitrogen purge flow, the transmission for both 60 and 400 Hz resolves to unity. This data corroborates the conclusion that a long-lived component of gaseous-phase (or very fine aerosol phase) ablation products appear to blanket th e target during ablation. This component is easily blown away from the ablati on area. This component does not have a rate effect, and is likely mitigated by plume evacuator s present on many clinical systems. Given the current findings, no rate dependen ce is expected over the range of 60 to 400 Hz examined here. Direct comparisons with prev ious plume dynamics studies ar e difficult, as each study uses a specific set of experimental conditions, namely fluence, wave length and laser beam diameter

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51 (spot size). However, the trends noted here are cons istent with the literature. The early images of Puliafito and co-workers show a similar plume evolution scheme with quicker dissipation than was noted here, which is expected due to the high er laser fluence used [69]. The time-scales are also very consistent with the plume velo cities on the order of 10 m/s reported by Hahn et al for an average (full-beam) fluence of 100 mJ/pulse [ 68]. Noack and co-workers reported that laser shielding by build-up of plume material is significant for large beams, noting that plumes generated by smaller-diameter laser beams, such as 1 mm, dissipate much quicker than their large-diameter counterparts [67]. The current stud y has found that laser shielding is insignificant for ablation with a 1-mm spot si ze at rates up to approximately 400 Hz. Pettit and Ediger found that the transmission through the ablation plume and excised cornea combined was minimized at 30 s at a value of 40% [79]. It is not expected that the reported transmission values match those determined here, as their probe wavelength wa s 355 nm and the pathleng th included the actual cornea itself, which is highly absorbing in the UV region. However, the evolution of transmission over time is consistent with the cu rrent findings. From their data, it appears that they also observed the lack of resolution of transmission to 100% at longer time scales.

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52 Figure 2-1. Experimental set-up fo r measuring ablation plume dynamics Figure 2-2. Schematic of th e transmission studies setup 193-nm Line Filter Optical Density Filters Lens 2 Scan Cube 193-nm ArF Lens 1 Laser 1 193-nm ArF Detector Mirror 1 Mirror 2 Scan CubeHomogenizer Pin Hole 193-nm ArF Pin Hole 193-nm Line Filter Optical Density Filters Pin Hole Detector Glass Quartz Eye Holde r Delay Generator Laser 3 To Oscilloscope To Oscilloscope Mirr o r 1 Mirr o r 2 L e n s 2 Scan Cube 193-nm ArF Lens1 Homogenizer Pin H o l e Eye Holder Cylindrical Lens Beam Dump Camera (ICCD) 532-nm Nd:YAG Delay Generator Delay Generator Laser 1 Laser2 Eye Holder ToPC 193-nm ArF

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53 Figure 2-3. Ablation plume images as a function of time following the ablating laser pulse. All images have the same intensity scale. 721 s 1.121 ms 121 s 421 s 1.021 ms 2.121 ms

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54 Figure 2-4. Long time scale ablation plume images All images have the same intensity scale. 0 2000 4000 6000 8000 10000 12000 0510152025303540Average Scattering Intensity (a.u.)Delay Time (ms) Figure 2-5. The average scattering intensity (ful l-image) of the ablation plume images as a function of time following the ablating lase r pulse with a 2 Hz repetition rate 2.5 ms 9 ms

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55 80 85 90 95 100 1101001000104Probe Laser Transmission (%)Delay From Ablating Laser ( m) Figure 2-6. ArF probe laser tr ansmission through the ablation plume as a function of time following ablation. Transmission was recorded at a fixed height of ~2 mm above cornea surface. Error bars are equal to one standard deviation. 90 95 100 105 110 0.11101001000104Probe Laser Transmission (%)Delay From Ablating Laser ( m) Figure 2-7. Transmission of ArF probe beam through non-ablating beam path. Error bars represent one standard deviation.

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56 Figure 2-8. Configuration of the nitrogen purge jet for the transmission loss experiment 0.90 0.95 1.00 1.05 60 Hz400 Hz60 Hz Flow400 Hz FlowProbe Laser Transmission (%) Quiescent AirCross Flow Figure 2-9. Average transmission of the ArF probe laser at a fi xed delay of 1.25 ms and fixed height of ~2 mm. Error bars are equal to one standard deviation. Probe ArF (GAM) Ablation ArF (Alcon) N2 jet 105 100 95 90

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57 CHAPTER 3 ABLATION PROFILES When varying surgical parameters, such as la ser repetition rate, it is essential that the geometry of the corneal resculpti ng remain the same. In this chapter, the possible impact of laser repetition rate upon ablation profiles is analyzed. Experimental Setup and Methods In all cases, the TUI (Santa Clara, CA) Ar F excimer laser output energy was nominally 2.7.0 mJ per pulse at the corneal eye plane and the spot size was approximately 1 mm. The laser is capable of producing lase r repletion rates from 1 Hz to above 400 Hz. Alcon proprietary software controlled the delivery of a specified refractive correction over a 6-mm treatment zone. As in clinical settings, the laser beam was oriented downward onto the treated material. Wax Ablation Profiles To ensure successful clinical outcome, it is crucial that the overall ablation profiles generated at the relevant laser repetition rates are statistically identical for a given refractive correction. The ablation depth gene rated by a particular system is a function of the incident laser fluence, with higher fluences pr oducing deeper ablation depths. In order to assess any variations in the pr ofiles generated by varying the laser repetition rate, corneal tissue ablation crat ers were analyzed. Two types of ablation were performed. For the initial set of experiments, a straight bore ablation was execut ed, where each successive shot was directed to the same spot on the cornea. A second set of experiments was completed using a scanning operation in which ablation craters were generated by superimposing a 25-shot sequence in the center of a 3-di opter, 6-mm zone ablation. The tr eatment was performed with the laser operating at rates of either 60 and 400 Hz using paired eyes from each bovine. Hence, one eye received the treatment at 60 Hz, and the se cond eye received the treatment at 400 Hz. Using

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58 superimposed shots on a standard profile provided a more clinical representation than a simple static laser beam because in clinical applications the time between laser pulses at a given location is significantly greater than the pulse-to-pulse firing time due to the scanning algorithm employed by the laser system. The system used to generate these patterns is sketched in Figure 3-1. The surgical 193-nm ArF excimer laser beam (Laser 1) passes through a beam homogenizer, a focusing lens and a pinhole before entering the scan cube. The scan cube contains two orthogonal mirrors that move in c oncert to generate a correction profile at the corneal plane. After exiting the scan cube, the beam then passes through a second focusing lens and two mirrors and is projected downward onto the corneal su rface. The bovine eye globe was held in a specially designed holder to keep it static and taut during the procedure. The intraocular pressure was not measured in this set of experiments; however, the weight of the top of the eye holder was used to keep a consistent pressure on all eyes. The purpose of keeping the eye surface taut was to prevent aberrations in the ablations from shriveling of the bovine eyes, which naturally occurs following excision. Not shown in the figure are two alignment lasers. The first is a diode laser that runs through the center of the cavity of Laser 1 and allo ws the beam of Laser 1 to be centered on the target (cornea). Th e second is a diode laser that is set to a height matching the ablation plane (the plane at which the spot of Laser 1 is minimized) and allows the target to be set in the ablation plane. In all experiments, whole bovine eye gl obes were extracted immediately following sacrifice (less than 20 minutes) and placed in se aled, buffered saline solution-filled plastic bags and kept at room temperature. Experiments were always performed within several hours following animal sacrifice. Prio r to all ablations, the cornea l surface was mechanically deepithelialized usi ng a scalpel edge.

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59 Immediately following completion of the abla tion, impressions of the craters were prepared using single drops of paraffin wax as previously documented [50]. Once the wax was thoroughly solidified, the impression was inverted a nd transferred to a microscope slide. A Zygo NewView white-light interferometer was then used to examine the impressions to obtain 3dimensional images of the full craters. The inte rferometer was outfitted with a 5X Michelson objective with a 0.5X magnification setting resulting in a net 2.5X surface view. Five eyes were analyzed at each laser repetiti on rate (10 eyes total). Figure 3-2 is a representative threedimensional model of a straight-bore ablation im pression. Figure 3-3 is a representative threedimensional model of a scanning ablation impressi on. At this stage, it vi rtually impossible to distinguish the two ablati on algorithms apart. In order to process the images, six 2-dimensi onal cross sections were extracted from each 3-dimensional interferogram using a star pattern such that each cr oss-section bisected the center of the ablation crater as shown in Figure 3-4 fo r a wax impression of a straight-bore ablation and Figure 3-6 for a wax impression of a scanning ablation. The correspondin g cross-sections are shown in Figure 3-5 and Figure 3-7, respectively. Figures 3-2 through 3-7 we re all obtained from the MetroPro software that accompanies the Zygo New View interferometer. The cross-sections were then exported from MetroPro as text fi les for further processing in Microsoft Excel. Parabolic trend lines, which provided good appr oximations of the corneal surface near the ablation site ( r2 > 0.9), were used to subtract the ove rall corneal surface curvature from each cross section, yielding the ablati on profiles. The necessity of this can be seen by examining Figure 3-5. There is an overall curv ature to the cross-section that is not negligible. Without this subtraction, the ablation profiles ob tained would be artificially low. Also, the cross-sections would be skewed in varying directions, as cen tering the ablation impression on the interferogram

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60 is difficult to do on a curved sample. The six subt racted cross sections were then combined and smoothed to yield the final ablation profile for each treated cornea. The parabolic trendlines represent the geometry of the cornea in the case of the straight-bore abla tions. In the case of the scanning ablations, the trendlines represent the overall 3-diopter myopic correction geometry, as the few millimeter field of view of the interfer ometer is less than the 6-mm correction zone. The overall steepness of the wax impr ession is less in Figure 3-7 than in Figure 3-5. This is the anticipated outcome, as a myopic co rrection (-3 diopters in this case) flattens the cornea in the ablation zone. Plastic Ablation Profiles Ablations were performed on PMMA samples as well as corneal samples. PMMA remains static post-ablation, eliminati ng the requirement of wax sa mples. Instead, white-light interferometry was performed directly on th e PMMA surface. This allows for cleaner interferometry images of the samples for thr ee reasons. First the wax samples contain voids associated with the solidification process, althoug h such effects were shown to be minimal [50]. Second, in the case of PMMA, interferometry is pe rformed on the sample directly, as opposed to an impression of the sample. This eliminates tran sfer errors associated with the mold process. Finally, the PMMA samples are flat, leading to clearer interferometry, as there is no drop-out associated with the linear height range of the instrument. As PMMA has been successfully used to model corneal ablation behavior, PMMA experime nts can be used to conf irm the results of the corneal ablation profile study, as well as to investigate other topics. The same experimental setup described in th e above section for corneal ablation profiles (Figure 3-1) was utilized for examining plas tic ablation profiles. The PMMA samples were placed on top of the eye holder, which was used to adjust the top surface of the polymer into the surgical (focus) plane. Once the ablations were performed, the PMMA samples were examined

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61 with white-light interferometry in the same mann er as the corneal samples. Both straight-bore and scanning ablations were performed on the PMMA samples. Figures 3-8 and 3-10 show representative three-dimensi onal scans of ablation craters on PMMA for straight-bore and scanning ablations, respectively. Figures 3-9 and 3-11 show the corresponding cross-sections for straight-bore and scanning ablations, respectively. The three-dimensional scans from PMMA can be compared with those from the wax impressions (Figures 3-2 and 3-3). The PMMA scans are much smoother as expected. One notable exception that differen tiates the PMMA analysis from the wax impression analysis is obvious from Figure 3-9. Since the surface of the PMMA is flat, parabolic trendlines for the overall geometry is no longer applicable. Thus, in the case of straight -bore ablation of the PMMA samples, linear trendlines were used. No te that the cross-sec tions in Figure 3-11 are similar to those in 3-9, as anticipated, as the same -3 diopter correction for myopia was performed in both cases. What sepa rates these two figures is the smoothness of the cross-sections in Figure 3-11. Imaged Ablation Profiles An entirely separate imaging technique was also developed implemented to determine corneal ablation profiles. The ablations were perf ormed in the same way as in the wax ablation studies, with the addition of two imaging cam eras. A Princeton iCCD camera (model 7489-0001) was used to center the curvature of the cornea in the direction orthogonal to the second camera. When the cornea is properly centered on this al ignment camera, the ablation will be performed on the apex of the cornea. This is essential to the measurements in these experiments. The second camera was used to record images of the corneal profile pre a nd post-operatively. The experimental setup is outlined in Figure 3-12.

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62 The concept of this technique is straightforwar d: a profile image of the cornea is taken with an Andor iCCD camera both preand post-operatively, and the pre image is subtracted from the post image. This difference between the two abla tion profiles is the abla tion profile. Each saved image was an average of 10 exposures. Figure 3-13 is an example image from the Andor camera. There is a clear definition betw een the eye (bottom section of the frame) and the surroundings (top section of the frame). In order to quantify this border, an edge finding technique was employed using MatLab. Text files from the Andor camera were manipulated using MatLab to determine the location of the corn eal surface. The individual vert ical pixel vectors have three distinct zones: a low flat line representing th e eye, a transition zone and a high flat line representing the surroundings. The low and high lines were averaged over several pixels to determine values for the eye and surroundings, respectively. These values were averaged, and the resulting value is considered the surface of th e eye. This operation is performed for each horizontal pixel location. Shown on Figure 3-13 is a dark line th at represents the edge found using this algorithm. The line is nicely consis tent across the entire profile. The MatLab codes used to achieve the edge profiles are in Appendi ces A and B. The ablations were performed at 92 Hz. Ablation Profile Results Wax Ablation Profiles The straight-bore ablation experiments will be discussed first, then the scanning ablation experiments. The straight-bore ablations of bovine corneal tissues were performed at nominally 3.0 mJ/pulse. The laser repetition ra te was varied while the number of shots remained a constant 20 shots. Ablations were performed on seven bovine eyes. Four ablations were performed on each eye. Each ablation was perf ormed on a separate corneal site one at each of the following laser repetition rates: 1, 10, 50 and 300 Hz. This experiment will ascertain whether or not the

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63 ablation rate (overall depth removed per laser puls e) varies with the laser repetition rate. Not every wax impression of the straight-bore ablation s was viable. In some cases, voids in the wax sample were located over the ablation impression. Four usable interferog rams were obtained for the 1 Hz samples, while three usable interfer ograms were obtained for each of the remaining laser repetition rates. Th e overall depths (the depth at the ce nter of the profile ) and corresponding standard deviations for each interferogram ar e listed in Table 3-1. The final ablation profiles obtained from each interferogram for each laser re petition rate were combined and smoothed to yield the final profile for each rate, as show n in Figure 3-14. The overall and corresponding standard deviations for each laser repe tition rate are list ed in Table 3-2. The width of the profiles in Figure 3-14 is cons tant as the laser repetition rate increases. Also, the overall ablation depth is also consistent acros s the profile. The profile created at 1 Hz is slightly deeper than that of th e other repetition rates. This is quantified in Table 3-2. This is likely due to unstable laser energy at 1 Hz, whic h is well below the intended operation range of the Alcon system. Finally, the average ablation rate (overall depth removed per laser pulse) was calculated to be 0.98 m/shot, based on the 10, 50 and 300 Hz data, which is consistent with Fisher & Hahn, 2004b. Scanning ablation experiments were also pe rformed on bovine corneas. This set of experiments had two purposes. First, ablations at 60 and 400 Hz were compared for potential rate effects. Second, a plume evacuator created by and borrowed from Alcon was temporarily put into place to determine if the ablation profile is affected by slight vacuum suction. The vacuum suction applied by the plume evacuator was so slight that it could not be felt, nor did it disturb tissue paper placed on the cornea. The intent of th e evacuator is to remove plume material from the ablation area in order to reduce the odor asso ciated with surgery. Five bovine corneas each

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64 were ablated using 60 Hz ablations and 400 Hz ablations without the plume evacuator in place. Three bovine corneas were ablated using 400 Hz ablations with the plume evacuator in place. All ablations were performed at laser energy of 2.7 mJ /pulse. As in the straight-bore ablations, not every wax impression was viable. In some cases, voi ds in the wax sample were located over the ablation impression. Two usable interferograms were obtained for the 400 Hz samples with vacuum applied, while for usable interferograms were obtained for both of the laser repetition rates without vacuum applied. The overall depths (the depth at the cente r of the profile) and corresponding standard deviations for each inte rferogram are listed in Table 3-3. The final ablation profiles obtained from each interferogram for each laser repetition rate were combined and smoothed to yield the final profile for each rate, as shown in Figure 3-15. The overall and corresponding standard deviations for each laser repetition rate and vacuum condition are listed in Table 3-4. The width of the profiles in Figure 3-15 is cons tant as the laser repetition rate increases with the number of shots. Also, the overall abla tion depth is also consis tent across the profile. This is quantified in Table 3-3. No differences in the profiles or ab lation depths are noted between 60 Hz and 400 Hz. Additionally, the presence of the plume evacuator does not appear to alter the ablation profile or overa ll ablation depth. This result is anticipated based on the plume imaging and transmission studies above, as the plume has developed beyond the immediate vicinity of the laser pulse. Fina lly, the average ablation rate (o verall depth removed per laser pulse) was calculated to be 0.95 m/shot, which is consistent with Fisher & Hahn, 2004b. Plastic Ablation Profiles The straight-bore ablation experiments will be discussed first, then the scanning ablation experiments. First, the number of shots applied in a single ablati on was varied as the number of

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65 shots remained a constant 1 Hz. Two ablations were performed for each of the following number of shots: 5, 10, 15, 20 and 25. This experiment w ill ascertain whether or not the ablation rate (overall depth removed per laser pulse) varies w ith the number of shots. The two final ablation profiles for each number of shots were combined a nd smoothed to yield the final profile for each number of shots, as shown in Figure 3-16. The overall depths (the depth at the center of the profile) are shown in Figure 3-17 with a linear tr endline applied for easy visual comparison. The equation of the linear regression line is y = 0.173x + 0.107 with an R value of 0.996. Finally, the ablation rates (overall depth removed per laser pu lse) were calculated as shown in Figure 3-18. It can be seen from the above thre e figures that the ablation rate ( m/shot) is a constant 0.18 m/shot ( =0.009) from 5-25 laser pulses at 1 Hz. No incubation effects are present after 5 pulses of the laser. Also, it is interesting to ex amine the growth of the ablation shown in Figure 16. The width of the profile is c onstant as the depth increases w ith the number of shots. The progression of the profil e is in the depth only. An Ophir Beamstar-FX-33 beam profiler was us ed to record the laser beam profile for comparison with the ablation profiles observed in the PMMA samples. Figure 3-19 shows the fluence profile (mJ/cm2) of the Alcon excimer laser beam recorded with Ophirs Beamstar program. The ablation profiles in Figure 3-16 and the fluence profile in Figure 3-19 can be combined to determine the ablation rate verses fluence acro ss the entire crater. In order to do so, as both plots are scatter graphs of disc rete data points, linear interp olation was utilized. The resulting data are shown in Figure 3-20. For the second straight-bore experiment performed, the number of pulses was kept constant at 25 shots, while the laser repetition rate was varied. Three ablations were performed

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66 for each of the following laser repetition rates: 1 Hz, 60 Hz, 230 Hz and 400 Hz. This experiment will ascertain whether or not the ablation rate (ove rall depth removed per laser pulse) varies with the laser repetition rate. The thr ee final ablation profiles for each number of shots were combined and smoothed to yield the final profile for each laser repetition rate, as shown in Figure 3-21. The overall depths (the dept h at the center of the profile ) are shown in Figure 3-22. First, it is interesting to note that the ablation profiles for 60, 230 and 400 Hz are indistinguishable from one another. However, at 1 Hz, the ablation profile is shallower than the other three. This observation is quantified by examination of the overall ablation depth. It can be seen from the figures that the ablation depth ( m/shot) is a constant 4.65 m ( =0.16) for 60 to 400 Hz for 25 shots. The ablation depth at 1 Hz, 4.33 m ( =0.1), is within two standard deviations of the mean depth for the othe r three rates. that the ablation depth ( m/shot) is a constant 4.57 m ( =0.22) for 1 Hz laser repetition ra tes for 25 shots. The corresponding ablation rate is 0.183 m/shot, which compares well with the ablation rate obtained from varying the number of laser pulses (0.18 m/shot). The ablation profiles in Figure 3-21 and the fluence profile in Figure 3-19 were also combined to determine the ablation rate ve rses fluence. The result is in Figure 3-23. After the straight-bore set of experiments were performed, experiments were performed using the scanning algorithm. The scanning experi ments have a dual purpose. First, ablations were performed at various rates to compare w ith the bovine eye studie s. Second, an issue was raised by Alcon regarding the ab lation algorithm. The original algorithm (stretched spiral) employs a counter-clockwise spiral ing flying spot that moves fr om the center of the cornea to the edge of the ablation zone. For some systems, the algorithm was reversed (reverse spiral) to employ a clock-wise spiraling fly ing spot that moves fr om the edge of the ablation zone to the

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67 center of the cornea. Given this change in algorithm, it was desirable to ensure that similar ablation results were obtained. PMMA ablation s were performed for each algorithm to study the problem. Three stretched spiral ablations and three reverse spiral algorithm ablations were performed for each of the following ablation rate s: 60 Hz, 92 Hz and 400 Hz. The same general analysis techniques used for the straight-bore abla tions above were used in these experiments as well. Figures 3-24 through 3-26 show the resu lting ablation profile results for each laser repetition rate. The stretched and reverse spiral algorithms in Figure 324 are nearly impossible to distinguish from one another. The stretched (S) and reverse (R) spiral algorithms in Figure 325 and 3-26 are identified w ith arrows for clarity. The overall ablation depths were compared to determine any rate effects or changes due to algorithm reversal. The average depths and standa rd deviations for each ablation (interferogram) are presented in Table 3-5. These results are pr esented in Figure 3-27 and Figure 3-28. Figure 327 is arranged for easy visual comparison of the stretched spiral and reve rse spiral algorithms. Figure 3-28 is arranged for easy visual comp arison of the laser repetition rates. From this data, it is easily concluded that the algorithm does not alter the overall ablation depth of the 9-diopter correction. Pa rticularly, this is very visible from the overlapping error bars in Figure 3-27. Also, the average overall ablation depth from all three laser repetition rates for stretched spiral and reverse spiral are 4.35 m ( =0.18) and 4.19 m ( =0.24). These values are within one standard deviation of each other. A students t-test was pe rformed for confirmation, and the samples may statistically be considered to have the same mean within a 95% confidence interval. Thusly, the stretched spiral and reverse spiral abla tion profiles were combined and

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68 smoothed to generate single ablation profiles a nd overall ablation depths for each of the three ablation rates, as shown in Figures 3-29 and 3-30. The overall ablation depth for ablations generated at 60, 92 and 400 Hz are 4.27 m ( =0.16 m), 4.36 m ( =0.18 m) and 4.17 m ( =0.28 m), respectively. This experiment confirms the result the scanning ablation expe riments conducted on bovine corneas; increasing the laser repetition rate up to 400 Hz does not a lter the ablation profile or the overall ablation depth. The ablation profiles and the fluence pr ofile in Figure 3-19 were also combined to determine the ablation rate verses fl uence. The result is in Figure 3-31. The ablation rates of Figures 3-20, 3-23 and 331 (nm/shot) are consis tent with the data from Srinivasan & Braren, 1989 depicted in Figure 1-7. At a fluence of 200 mJ/cm2, the ablation rate obtained from the PMMA experiments pres ented in this section is approximately 90 nm/shot, while the published data indicates a rate of approximately 250 nm/shot. It is unclear if the graphed fluence in Figure 1-7 is the peak flue nce or the average fluence. The average fluence is generally half that of the peak fluence, which would explain differe nce between the values. Additionally, one can determine the threshol d fluence for PMMA as approximately 25 mJ/cm2, which is in agreement with th e value of approximately 35 mJ/cm2 obtained from Figure 1-7 [49]. Imaged Ablation Profiles Figures 3-32 and 3-34 are examples of preablation iCCD images. Figure 3-33 is the postablation profile of the same eye as that of Fi gure 3-32. Likewise, Figure 335 is the post-ablation profile of the same eye as that of Figure 3-34. The edge as found by the MatLab program in Appendix A is also diagrammed in the figures. The axes of the figures are pixels, and each pixel represents 5.1 microns.

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69 Seven paired bovine eyes were analyzed. From each bovine, one eye was ablated using the stretched spiral pattern, and the second wa s ablated using the re verse spiral pattern. Representative ablation profiles for each algorith m are shown in Figure 3-36. For analysis, first, the pixel numbers of the reverse spiral pattern was subtracted fr om those of stretched spiral pattern. Using the calibration of 5.1 m/pixel, the number of pixels composing the difference was converted to actual dimensi ons. Figure 3-37 is a representati ve differential ablation profile. Table 3-5 contains the maximum ablation depth for each bovine eye and ablation algorithm. At the bottom of the table, the averages and sta ndard deviations for each algorithm are listed. The maximum ablation depths ar e nearly identical (164.62 and 163.55 m, for stretched spiral and reverse spiral, respectively), and the standard deviations ar e quite large (31.14 and 21.91 m, for stretched spiral and reve rse spiral, respectively). At this juncture it is difficult to tell any difference between the ablations generate d by the stretched spiral and the reverse spiral algorithms. For further analysis, the differential ablation profiles of all seven eyes were then averaged for a final average profile. Lastly, ten pi xels at each end of th e profiles were averaged and the slope between them was us ed to subtract a baseline from the averaged profile. The final result is presented in Figure 3-38. The differential ablation profiles show a clear pattern, similar to that of each individual ablation. However, the precision of this method is sti ll an issue. It is very difficult to line up the cornea such that it is exactly centered on the tw o iCCD cameras. This challenge explains the large variation in the ablation depths, as slight translation off of the apex of the cornea will significantly alter the measured ab lation depths. However, failure to achieve sufficient alignment does not affect the trend of the ablation profile trend. As such, it is impo ssible to clearly state (using this method) whether or not algorithm reversal has an e ffect on the ablation geometry.

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70 Ablation Profile Summary The main results come from the wax impressions and PMMA studies. From these experiments, we can clearly state that the lase r repetition rate does not significantly alter the geometry of the ablation profile The width of the ablation prof iles in bovine eyes was found to be constant as the laser repetiti on rate increases and as the number of shots increases. Also, the overall ablation depth is also c onsistent across the profile for va rying laser repetition rates. The average bovine cornea ablation rate (overall depth removed per laser pulse) was calculated to be 0.98 m/shot, in the straight bore experiments and 0.95 m/shot in the scanning experiments. The PMMA experiments indicate that reversi ng the ablation algorithm does not alter the ablation geometry. These experiment s also concluded that there is no statistical difference in the ablation profiles as the laser repe tition rate is increased from 60 to 100 Hz. The ablation rate for PMMA was found to be approximately 0.17 m/shot. It is very important to note here that this rate cannot be compared to the bov ine cornea rate. In the literature review, it was pointed out that a conversion of 1.8 m of corneal tissue is removed per 1.0 m of PMMA. The laser was tuned by Alcon between the bovine corn eal studies and the PMMA studies At that time, the fluence profile of the laser beam wa s changed from maxing out at approximately 650 mJ/pulse to approximately 475 mJ/pulse. One would anticipate that at an equal la ser output energy, the ablation depth created by the lase r to decrease with decrease in peak fluence. Thus, it is not unexpected that the PMMA ablation rate relativ e to the corneal ablation rate is lower than expected based on the literature. Additionally, the molecular weight of PMMA is known to affect the mechanism of PMMA ablation, a nd thus the ablation ra te [80]. This is partially responsible for the large range of reported PMMA thresholds, as well. Additionally it was determined that, in PMMA, reversal of the ablation algorithm doe s not affect the geometry of the ablation.

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71 Figure 3-1. Experimental set-up for ablation of bovine corneas Figure 3-2. White light interferom etry 3-dimensional profile of a wax impression of a straightbore ablation crater Eye Holder Mirror 1 Mirror 2 Lens 2 Scan Cube 193-nm ArF Lens 1 Homogenizer Pin Hole Laser 1

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72 Figure 3-3. White light interfer ometry 3-dimensional profile of a wax impression of a scanning ablation crater Figure 3-4. White light interferom etry 2-dimensional profile of a wax impression of a straightbore crater

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73 Figure 3-5. White light interfer ometry 2-dimensional cross sec tions of a wax impression of a scanning crater Figure 3-6. White light interfer ometry 2-dimensional profile of a wax impression of a scanning crater

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74 Figure 3-7. White light interfer ometry 2-dimensional cross sec tions of a wax impression of a scanning crater Figure 3-8. White light interferom etry 3-dimensional profile of a straight-bore ablation crater on PMMA

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75 Figure 3-9. White light interferom etry 2-dimensional cross secti ons of a straight-bore ablation crater on PMMA Figure 3-10. White light interferometry 3-dimensional profile of a scanning ablation crater on PMMA

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76 Figure 3-11. White light interf erometry 2-dimensional cross sections of a scanning ablation crater on PMMA Figure 3-12. Experimental set-up for imaging ablation plume profiles Lens 2 Mirror 1 Mirror 2 Scan Cube 193-nm ArF Lens1 Homo g enizer Pin Hole Alignment Princeton Camera (ICCD) Laser 1 Imaging Andor Camera (ICCD) E y e Holde r To PC To PC

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77 Figure 3-13. Representative profile image of a bovine eye showing the cl ear definition between the eye and the environment. Both axes ar e camera pixel numbers. An edge profile is also shown. Table 3-1. Overall ablation depths ( m) for bovine straight-bore ablations for each interferogram for various laser repetition rates 1 Hz 10 Hz 50 Hz 300 Hz Average Standard deviation Average Standard deviation AverageStandard deviation AverageStandard deviation 23.06 3.88 21.70 2.1921.122.4317.930.77 19.88 2.07 16.76 3.3519.751.3022.003.46 24.30 1.51 20.66 0.5420.831.5316.961.55 21.11 4.04

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78 -25 -20 -15 -10 -5 0 5 -1.5-1-0.500.511.5 1 Hz 10 Hz 50 Hz 300 HzAblation Depth ( m)Radius (mm) Figure 3-14. Straight-bore ablati on crater profiles created on bovi ne corneas using 20 shots at various laser repetition rates Table 3-2. Overall ablation depths ( m) for bovine straight-bore ablations for each laser repetition rates Rate Average Standard deviation 1 Hz 22.18 3.33 10 Hz 19.50 3.07 50 Hz 20.44 1.81 300 Hz 18.91 3.07 Table 3-3. Overall ablation depths ( m) and standard deviations for bovine scanning ablations for each interferogram for various laser repetition rates Without evacuator With evacuator 60 Hz 400 Hz 400 Hz Average Standard deviation Average Standard deviation Average Standard deviation 22.49 1.74 24.79 4.2323.851.17 20.95 1.58 20.95 0.9924.482.34 26.08 2.44 26.78 1.13 24.71 2.10 23.58 1.74 23.76 0.77 19.47 2.03

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79 -25 -20 -15 -10 -5 0 5 -1.5-1-0.500.511.5 60 Hz 400 Hz 400 Hz VacuumAblation Depth ( m)Radius (mm) Figure 3-15. Scanning ablation crater profile s created on bovine corneas using 25 shots at various laser repetition rates Table 3-4. Overall ablation depths ( m) for bovine scanning ablati ons for each laser repetition rates Rate Average Standard deviation 60 Hz 23.642.47 400 Hz 23.163.37 400 Hz ( with e vacuator ) 24.071.79

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80 -5 -4 -3 -2 -1 0 1 -2-1.5-1-0.500.511.52 5 Pulses 10 Pulses 15 Pulses 20 Pulses 25 PulsesAblation Depth ( m)Diameter (mm) Figure 3-16. Ablation profiles in PMMA created at 1 Hz for va rious numbers of laser pulses 0 1 2 3 4 5 051015202530 y = 0.10669 + 0.17309x R= 0.99583 Overall Ablation Depth ( m)Number of Laser Pulses Figure 3-17. Ablation depths ( m) in PMMA created at 1 Hz for various numbers of laser pulses. The error bars repres ent one standard deviation.

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81 0 0.05 0.1 0.15 0.2 0.25 510152025Ablation Rate ( m/pulse)Number of Laser Pulses Figure 3-18. Average ablation rates ( m/shot) in PMMA created at 1 Hz for various numbers of laser pulses. The error bars repr esent one standard deviation. 0 100 200 300 400 500 600 -1-0.500.51Fluence (mJ/cm2)Radius (mm) Figure 3-19. Fluence profile of the Alcon laser beam in mJ/cm2

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82 -50 0 50 100 150 200 0100200300400500600 5 Shots 10 Shots 15 Shots 20 ShotsAblation Rate (nm/shot)Fluence (mJ/cm2) Figure 3-20. PMMA ablation rate (n m/shot) verses laser fluence (mJ/cm2) for various numbers of laser pulses -5 -4 -3 -2 -1 0 1 -2-1.5-1-0.500.511.52 1 Hz 60 Hz 230 Hz 400 HzAblation Depth ( m)Radius (mm) Figure 3-21. Ablation profiles in PMMA created with 25 laser pul ses for various laser repetition rates

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83 0 1 2 3 4 5 160230400Overall Ablation Depth ( m)Laser Repetition Rate Figure 3-22. Ablation depths ( m) in PMMA created with 25 laser pulses for various laser repetition rates. The error bars represent one standard deviation. -50 0 50 100 150 200 0100200300400500600 1 Hz 60 Hz 230 Hz 400 HzAblation Rate (nm/shot)Fluence (mJ/cm2) Figure 3-23. PMMA ablation rate (n m/shot) verses laser fluence (mJ/cm2) for various laser repetition rates

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84 -5 -4 -3 -2 -1 0 1 -1-0.500.51 60 S 60 RAblation Depth ( m)Distance (mm) Figure 3-24. PMMA stretched spiral (S) and re versed spiral (R) abla tion profiles at 60 Hz -5 -4 -3 -2 -1 0 1 -1-0.500.51 230 S 230 RAblation Depth ( m)Distance (mm) Figure 3-25. PMMA stretched spiral (S) and re versed spiral (R) abla tion profiles at 230 Hz R S

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85 -5 -4 -3 -2 -1 0 1 -1-0.500.51 400 S 400 RAblation Depth ( m)Distance (mm) Figure 3-26. PMMA stretched spiral (S) and re versed spiral (R) abla tion profiles at 400 Hz Table 3-5. PMMA ablation depths ( m) and standard deviations ( m) for the stretched spiral and reverse spiral algorithms a nd ablation rates of 60, 92 and 400 Hz Ablation Rate Pattern Average Standard deviation A1 60 Stretched 4.250.19 A2 60 Reverse 4.280.20 A3 60 Stretched 4.300.09 A4 60 Reverse 4.280.21 A5 60 Stretched 4.250.18 A6 60 Reverse 4.280.15 A7 92 Stretched 4.600.09 A8 92 Reverse 4.380.11 A9 92 Stretched 4.380.12 A10 92 Reverse 4.160.20 A11 92 Stretched 4.320.07 A12 92 Reverse 4.310.17 A13 400 Stretched 4.420.15 A14 400 Reverse 4.220.19 A15 400 Stretched 4.310.18 A16 400 Reverse 3.970.20 A17 400 Stretched 4.280.23 A18 400 Reverse 3.850.25 S R

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86 0 1 2 3 4 5 6 6092400 Stretched ReverseOverall Ablation Depth ( m)Laser Repetition Rate (Hz) Figure 3-27. PMMA ablation depths ( m) for the stretched spiral and reverse spiral algorithms at each ablation rate. Error bars represent one standard deviation. 0 1 2 3 4 5 6 StretchedReverse 60 Hz 230 Hz 400 HzOverall Ablation Depth ( m)Ablation Algorithm Figure 3-28. PMMA ablation depths ( m) for 60, 230 and 400 Hz for each algorithm. Error bars represent one standard deviation.

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87 -5 -4 -3 -2 -1 0 1 -1-0.500.51 60 Hz 230 Hz 400 HzAblation Depth ( m)Distance (mm) Figure 3-29. PMMA combined abla tion profiles for 60, 230 and 400 Hz 0 1 2 3 4 5 6092400Overall Ablation Depth ( m)Laser Repetition Rate (Hz) Figure 3-30. PMMA combined overall ablation depths ( m) for 60, 230 and 400 Hz. Error bars represent one standard deviation. 400 Hz 60 Hz 230 Hz

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88 -50 0 50 100 150 200 0100200300400500600 60 Hz 400 Hz 230 HzAblation Rate (nm/shot)Fluence (mJ/cm ) Figure 3-31. PMMA ablation rate (n m/shot) verses laser fluence (mJ/cm2) for various laser repetition rates Figure 3-32. Representative preablation profile image of a bovine eye. Both axes are camera pixel numbers. The edge profile is also shown.

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89 Figure 3-33. Representative postablation profile image of a bovine eye. Both axes are camera pixel numbers. The edge profile is also shown. Figure 3-34. Representative preablation profile image of a bovine eye. Both axes are camera pixel numbers. The edge profile is also shown.

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90 Figure 3-35. Representative postablation profile image of a bovine eye. Both axes are camera pixel numbers. The edge profile is also shown. 0 50 100 150 200 -3.00-2.00-1.000.001.002.003.00 Stretched ReversedAblation Depth ( m)Position (mm) Figure 3-36. Representative stretched spiral and reverse spiral ablation profiles for a paired set of eyes

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91 -30 -20 -10 0 10 20 30 0100020003000400050006000Ablation Depth ( m)Diameter ( m) Figure 3-37. Representative differe ntial ablation profile (stretched spiral minus reverse spiral) of a bovine eye. This differential profile is generated from the eyes in Figures 3-33 through 3-35. Table 3-6. Maximum ablation depth for pair ed stretched and reve rse spiral ablation Stretched spiral Reverse spiral 222.08 150.39 182.29 157.83 120.21 169.43 186.81 139.85 150.61 199.01 143.04 145.22 147.87 153.67 160.48 192.98 195.42 249.49

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92 -5 0 5 10 15 20 25 0100020003000400050006000Ablation Depth ( m)Distance ( m) Figure 3-38. Average differential ablation profile (stretched spiral minus reverse spiral) image of a bovine eye created at a lase r repetition rate of 92 Hz

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93 CHAPTER 4 HISTOLOGY The previous chapters have focused on the efficacy of the tissue removal. They sought to verify that changing the laser repe tition rate of the surgical lase r in corrective refractive surgery does not physically a lter the ablation process. It must sti ll be checked that the underlying tissue has no additional adverse damage as compared to current clinical rates. As there is less time between laser pulses for thermal energy to disperse, there is potential for an increase in tissue damage. Since the wound healing process is comple x, and much research has been devoted to the topic, it is essential that incr easing the laser repetition rate doe s not induce additional injury. Experimental Setup and Methods To assess any histological changes to the underlying stroma caused by varying the laser repetition rate, corneal ablations were performe d over a 6-mm zone with a standard 9-diopter correction, using the experimental set-up of Figur e 2-1. This is the maximum correction typically applied for this surgical system, thus any addi tional harm to the underlying tissue would be maximized by using this algorithm. Ablations were performed using laser repetition rates of 60, 230 and 400 Hz. For these experiments, no additional laser shots were added in the center as was done in the ablation profile study described above. For assessment of rate effect s, conventional microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (T EM) were performed on corneal buttons (four at each rate), which were immediately harveste d using 8-mm biopsy punches. For conventional microscopy, the buttons were dehydra ted in a graded seri es of alcohol solutions. They were then cleared with xylene solution and fixed into a paraffin bl ock. Corneal samples of 4m thickness were sectioned and stained with H&E (hematoxy lin and eosin). Light microscopy images were processed and stored for each sample. For TE M and SEM analysis, corneal buttons (four for

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94 each rate) were harvested and fixed in 2.5% glutarlaldehyde in 0.1 M cacodylate buffer and prepared for analysis. The cornea buttons were bisected and postfixed in 2% osmium tetroxide for two hours. Small pieces of the cornea were embedded in low-viscosity epoxy medium, thin sectioned, stained with uranyl acetate and lead citrate, a nd viewed with a JEOL 100 CX transmission electron microscope. The other half of the cornea was prepared for SEM by critical point drying. The tissue was glue d to stubs, sputter coated with gold palladium, and viewed with a JEOL 25 CF scanning electron microscope. SEM and TEM images were then processed and stored for each sample. These sections were prepared and microscopy was performed by Dr. Henry Edelhausers team at Emory University SEM and TEM samples were analyzed at two separate locations, the ablation cr ater side and the ablation crater floor, as shown in Figure 4-1. Histology Results Conventional microscopy was performed first. F our eyes were ablated each with 60 Hz and 400 Hz. For each laser repetition rate, one eye was given a 3-diopter correction, one eye was given a 6-diopter correction, and two eyes were given a 9-diopter correction. Figure 4-2 shows representative high magnification optical microscopy images of th e ablated corneas. The section on the right is from a cornea ablated at 60 Hz, wh ile the section on the right is from a cornea ablated at 400 Hz. The arrows on the image point to the surface that was ablated. Visible in the bottom of these images is Descemets membrane and the endothelial layer. Descemets membrane separates the stroma, whic h composes the majority of the section, from the dark thick endothethial layer. Note that th e epithelial layer and Bowmans membrane are not present. The epithelial layer wa s mechanically removed prior to ablation, and Bowmans layer was removed by the ablation. Qualitatively, no di fferences were observed between the samples generated at 60 Hz and t hose generated at 400 Hz.

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95 For further evaluation of potential differe ntial damage, SEM and TEM images were analyzed. Four eyes were ablated each at 60 Hz and 400 Hz with 9-diopter corrections, the maximum correction clinically applied by the Alcon laser system. To provide higher magnification, SEM images of the extracted corn eas ablated at 60 and 400 Hz were generated and are shown in Figure 4-3 at various magnifi cations. When comparing similar conditions for the image pairs, both ablation conditions (60an d 400-Hz laser repetition ra te) appear to present similar features, suggesting that no differentia l response exists betw een the two ablation conditions. In order to quantitatively assess any ablation rate effects, TEM anal ysis was performed to directly image the collagen fibrils. TEM images of corneas ablated at 60 and 400 Hz are shown in Figure 4-4 at the same magnification for dir ect comparison. Just as with the SEM images, no additional tissue damage is visually noticeable, including no visually obs ervable condensing or distortion of collagen fibrils. The TEM analysis revealed no differential damage between the two ablation rates based on the observed structures. In order to quantify information that ma y be represented in the TEM images, the configuration of collagen fibrils at the ablation surface was examined more closely. The TEM images (three eyes for each rate) were broken in to squares normal to the ablated crater surface, and the number of collagen fibrils (i.e. dots) in each square was counted. The counts (corrected for varying magnifications) were then comp ared. At 60 Hz, an average of 221 fibrils/ m2 ( = 36 fibrils / m2) were observed adjacent to the ablation surface. At 400 Hz, an average of 263 fibrils/ m2 ( = 22 fibrils/ m2) were observed. For a 95% confid ence value (t-test), there is no statistical difference in collagen fibril densit y near the ablation surface between 60 and 400 Hz ablation rates.

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96 Histology Summary Based on conventional microscopy, no differen ces were observed between the samples generated at 60 Hz and those generated at 400 Hz. In the SEM samples, when comparing comparable conditions for the image pairs, bo th ablation conditions (60and 400-Hz laser repetition rate) appear to presen t similar features, suggesting that no differential response exists between the two ablation conditions. In the TEM images, just as with the SEM images, no additional tissue damage is visually noticeable, including no visually obs ervable condensing or distortion of collagen fibrils. The TEM analysis revealed no differential damage between the two ablation rates based on the observed structures. Fo r a 95% confidence value (t-test), there is no statistical difference in collagen fibril densit y near the ablation surface between 60 and 400 Hz ablation rates. Overall, the collagen fibrils do not show any signs of disorganization, size changes or coagulation. Based on the microsc opy analysis, it is concluded that no thermal damage or differential thermal damage (i.e. 60 vs. 400 Hz) is observed under these ablation conditions.

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97 Figure 4-1. Location guide for SEM and TEM microscopy samples Figure 4-2. High magnificat ion H&E-stained microscopy images of the ablation crater for laser ablations created at 60 Hz and 400 Hz. Location Guide Ablation Crater Side Ablation Crater Floor 60 Hz 400 Hz

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98 Figure 4-3. SEM images of ablation craters cr eated at 60 Hz and 400 Hz A) Overall ablation crater. B) Ablation crater side s. C) Ablation crater floor. A B C

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99 Figure 4-4. TEM images of bovine cornea cross-s ections following laser ablation at 60 Hz and 400 Hz. The upper surface is the ablation surf ace, and the individual collagen fibrils are visible. The scale bar is 280 nm. 280 nm 280 nm 60 Hz 400 Hz

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100 CHAPTER 5 SUB-ABLATIVE PERTUBATIONS The work presented in this res earch proposal thus far has been of a more practical nature. However, the fundamental mechanism of tissue removal in corneal refractive surgery remains unresolved. The research presented here seeks to better understand the underlying process of corneal photoablation. Current th eories suggest that below th e ablation threshold, the energy deposition by the excimer laser into the corneal tissue results in a solely photothermal reaction, and that photochemical reactions only begin to o ccur once the threshold has been breached [5]. This experiment will examine laser-induced cha nges in collagen and amino acid solutions at subablative conditions. Experimental Setup and Methods Transmission experiments were implemented to study the reaction of collagen and amino acid solutions to sub-ablative lase r fluences. A sub-abla tive laser beam is used to perturb the solution sample, and a lower energy probe beam is used to determine the transmission through the sample. The change in transmission can be recorded for each pulse of the perturbing laser and analyzed to determine changes in the sample due to the sub-ablative perturbing laser pulse. In order to investigate the role of photochemical ef fects at low fluences, two sample types were analyzed. The first is a solution of calf-skin Type II collagen dissolved in acetic acid. The second is a solution of the three cons tituent amino acids (glycine, pr oline and hydroxyproline) in acetic acid. The latter solution is an approximation of the collagen solution without the peptide bond, noting that the peptide bond is be lieved to be the primary chro mophore of corneal collagen in laser photorefractive surgery [5, 14] If energies less than the th reshold value result in solely photothermal reactions within the tissue, the re sults obtained from the two solutions should be

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101 comparable. If the response of the two soluti on types gives different results, then one may conclude that the peptide bond pl ays a definite role in the s ub-ablative reaction of corneal collagen to excimer laser pulses, pointi ng to a distinctive photochemical role. These experiments were performed using alte rnately a 193-nm excime r laser or a 355-nm Nd:YAG laser as the sub-ablative perturbation sour ce. In both cases, the 193-nm excimer laser was used as the probing source. In the case of the 193-nm pert urbation source, the GAM excimer laser acts in a dual role. Specifi cally, the GAM laser will be us ed to both perturb the collagen sample with a sub-ablative fluence and to probe the transmission through the sample, as shown in Figure 5-1. In the case of the 355-nm pert urbation source, the Big Sky frequency tripled Nd:YAG laser (355 nm) perturbed the sample, while the GAM excimer laser acted as the probe beam only. In both cases, the GAM laser beam is immediately passed through an aperture, which passes a circular, central section of the elongated beam to a quartz flat. The flat allows 95% of the beam to transmit through it, while reflecti ng the remaining 5% of the beam. The higher energy portion may be used as the perturbation b eam, and the lower energy portion is the probe beam. For 193-nm experiments, the perturbation beam is turned at a 90o angle and passes through the sample cell, which is detailed in Figure 5-3. For 3 55-nm experiments, this higher energy excimer beam is blocked with a beam dump, and the Nd:YAG laser beam is reduced with an aperture, then turned at 90o and passed through the sample cell. The 193-nm probe beam passes through 4 mirrors on its path to the sample cell. The length of this path is designed to generate a delay relative to the 193-nm pertur bation beam. Approximately one nanosencond of delay is imposed for every foot of beam travel The optical delay path was designed to achieve 20 ns of pulse separation between the perturbing and probe laser pulses. This was necessary in order to distinguish the probe pulse from th e perturbing pulse. Also, it ensured that the

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102 perturbing pulse has ended prior to the start of the probing pulse, as the full pulse-width of the GAM laser is approximately 20 ns. In the case of 355-nm perturbation, the delay was induced using delay generators. The delayed probe beam is collimated and co-incident with the perturbing beam as they pass through the sample cell. Fast phototubes (~2 00 ps rise time) were used to collect the incident and transmitted light Optical density filters were used to ensure signal linearity, and 193-nm narrow line filters were used to elim inate fluorescence signal. The detectors were connected to a LeCroy Waverunner oscilloscope, which was used to record the incident and transmitted spectra. The transmission measurements were taken with two different timing methods. In the first, a perturbation shot was applied, and then the in cident and transmitted spectra are recorded. This was repeated ten times, and the sp ectra were averaged. This was then repeated in intervals, with one shot between each, up to 99 shots total. This is referred to as the sy nchronized method. In the second, ten perturbation shots were applied to the sample, and then an average of ten incident and transmitted probe spectra was recorded. This al so was then repeated in intervals, with one shot between each, up to 99 shots total. This is referred to as the unsynchronized method. The first method allows any dynamic processes that occur on the order of nanoseconds to influence the transmission signal. In the se cond, several seconds transpire between the perturbation and the transmission probe measurements, thusly any pr esent dynamic processes are expected to have resolved, and the static (or perm anent) results will be obtained. Both collagen and amino acid solutions were analyzed in the sample cell. Collagen Type II derived from calf skin was used to generate th e collagen solution by combining 1 mg of collagen per 1 ml of 0.5 N acetic acid. The amino acid solu tions were created by placing equal parts of 100 mg each of the amino acids glycine, prol ine and hydroxyproline per 30 ml 0.5 N acetic acid.

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103 The amino acid solution is essentially equal to the collagen solution le ss the peptide bonds. In addition, the amino acid solution is ten times more concentrated than th e collagen solutions in order to boost the transmission si gnal, as they are lacking the anticipated primary chromophore, peptide bonds. The incident probe beam signal was compared to the transmitted probe beam signal to determine the transmission, In general terms, the transmission is defined as the following. 0) ( I I (5-1) In this equation, I() is the intensity of the laser light after passing through the solution path length, and I0 is the incident laser intensity. Give n the Beer-Lambert law, the absorbance may be calculated from the transmission. L Ne (5-2) L N A 434 0 (5-3) In the above equations, N is the number density of absorbers (cm-3), is the absorption crosssection of the absorbers (cm2), L is the pathlength of the sample (cm), and A is the absorbance of the sample. The total change in transmission ma y be a result of contributions by both the acetic acid and the collagen or amino acids, such that the total transmission is the product of the transmission due to collagen and the transmi ssion due to acetic acid. The absorbance of the collagen or amino acids may be decoupled from acetic acid and calculated from the following equation. ) ln( lncollagen aceticacid totalA (5-4) In order to obtain the transmission of acetic acid only, the experiments were also performed with 0.5 N acetic acid in the sample cell. Once the ab sorbance is calculated, the number density of

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104 absorbers may be calculated, as the absorption cross-section and pathlength are known. The absorption cross-section is 1.19 E -17 cm2 per amino acid unit [8]. Other known values include the number of bonds per collagen macromolecule (3,465) and the average atomic mass per macromolecule (308,000 g/gmol) [8]. The number of absolute absorbers may be calculated from the number density by multiplying by the sample volum e. In all cases, the diameter of the probe beam is 2.6 mm. For 193-nm perturbations, experiments were performed with 0.55, 0.77, 0.93 and 1.05 mJ/pulse energy. Figure 5-4 shows a re presentative cross-section of the 193-nm perturbation beam. The beam has a top hat prof ile. The bounds of the probe beam have been drawn on the Figure to indicate that the probe beam diameter is less than that of the perturbation beam. In summary, the number of absorbers may be calculated from the transmission measurements. The perturbing pulse had an energy range fr om approximately 0.5 to 1.2 mJ/pulse over a approximately 2.6-mm beam resulting in a fluence 9 to 21 mJ/cm2, while the probing pulse had a fluence of less than 0.5 mJ/cm2. For 355-nm perturbations, expe riments were performed with 0.48 and 1.2 mJ/pulse energy. For each energy, three films of sample solution (collagen or amino acid) were analyzed along with three films of acetic acid solution, with four spots on each, for a total of 12 experiments. The number of laser pul ses converts into number of cumulative photons using the relation following relation: c h h Ep (5-5) In the above equation, Ep is the photon energy, h is Plancks constant, is the frequency of the laser light, c is the speed of light, and is the wavelength of the la ser. The total pulse energy, ET, can then be divided by the photon energy to ge t the number of photons per laser pulses, n.

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105 p TE E n (5-6) Therefore, the number of absorbers may be calcula ted with respect to the number of incident photons. Sub-Ablative Results The results are presented in tw o sections. The 193-nm excimer pe rturbation resu lts will be discussed first, followed by the results of 355-nm pert urbation results. Th e results will be discussed with respect to each other in the summary section to follow. 193-nm Perturbations An example of the raw incident and transmitted signal spectra obtained from the oscilloscope is shown in Figure 5-5. In the figure, the peak-to-p eak pulse separation is 18 ns. As this separation is due to the physi cal delay path, there is no jitter in the time separation. In order to convert this data into transmission, the transm itted and incident probe beam signals were first summed, then the background signal was subtracted from the sums, and the ratio of the two was taken. Using Equation 5-4, the absorbance for each sample was calculated for each time increment and pulse energy for both timing sc hemes (synchronized and unsynchronized). The average absorbance values of all twelve repetiti ons for each condition are presented in Table 5-1 with their corresponding standard deviations for collagen soluti ons. The results for amino acid solutions are presented in Table 5-2. It is noted that the reference acetic acid signal did not vary with number of shots applied, and was constant for each perturbation energy. From looking at these two tables, two trends ar e observed. First, the absorbance decreases with the number of perturbation shots for both the synchronized and unsynchronized cases. Second, the absorbance values are less for the synchronized case than the unsynchronized case.

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106 To further analyze the data, the extinction coefficients (N ) were calculated by dividing the absorbance by the pathlength of 0.0084 cm. This pathlength was determined by plugging in the average collagen transmission value of appr oximately 0.5 into Equation 5-2 along with the values of (1.19E-17 cm2) and N (6.77E18 cm-3) for the amino acid unit (amino acid + peptide bond). This value of N is calculated from publishe d values of the molecular mass of an amino acid unit (308,000 g/mol) and the number of pe ptide bonds per collagen macromolecule (3,465). The constant values were obtained from the wo rk of Fisher and Hahn [8]. This calculated pathlength is consistent with physical measurements of the collagen cells. The calculated extinction coefficients have b een tabulated for both timing variations and all energies with respect to th e number of incident photons as cal culated using Equations 5-5 and 5-6. Table 5-3 includes all values for collagen so lutions, while Table 5-4 includes all values for amino acid solutions. In order to further examine the trends in the data, the extinction values have all been plotted (Figures 5-6, 5-7, 5-8 a nd 5-9 for collagen solutions; Figure 5-10 for amino acid solutions). For collagen solutio ns, there is a decrease in ex tinction coefficient with photons, but the extinction coefficient remains constant for amino acids. For further calculations, the average amino acid extinction co efficient for synchronized (43.58, =1.44) and unsynchronized series (54.82, =1.66) will be used. In both solutions, there is a clear o ffset between the synchronized and unsynchronized cases, which i ndicates the presence of a dynamic component. The overall downward trend of the unsynchronized da ta confirms that a steady-state change is induced in the collagen solutions. The dynamic component was first analyzed in th e context of the ami no acid solutions. The change in extinction coefficient with respect to incident photons may be calculated from the following equation:

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107 n K K dn dKsync unsync (5-7) In the above equation, dn dK is the change in extinction coe fficient with respect to incident photons, K is the average extinction coefficient and n is the number of photons per pulse. The value obtained for dn dK is -1.24E-14 cm-1/photon, =2.43E-15. The number density of absorbers (1.41E20 amino acid per cm3, =8.68E18) is also constant and was calculated by dividing by the absorption cross-section of 4.74E-19 cm2 for the amino acids [8]. Lastly, the number of absorbers (2.56E23 amino acid units, =1.95E22) was calculated by multiplying the density by the sample volume of 0.445 mm3. The extinction coefficients of the unsynchroni zed collagen series are analyzed next. From this data, the number of peptide bonds may be calculated at each time step by resolving the overall extinction coefficient into its peptid e bond and amino acid contributions using the following equation: AA AA p p AAU AAU AA p unsync extN N N K K K (5-8) In the above equation, the subs cripts AAU, P and AA are used to denote amino acid unite, peptide bond and amino acid, respectively. This e quation can be rearranged to the following: 96 0 04 0AA AAU AAU P AA AAU AA AAU PN N N N N (5-9) The absorption cross-section ratios for amino acids and peptide bonds are known as 0.04 and 0.96, respectively [8]. The number density of the amino acid units per each time step is easily calculated from the overall extinction coefficient by dividing by the absorption cross-section of

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108 1.19 E -17 cm2. The number density of the amino acids is constant, as seen from the amino acid experiments that there is no stea dy-state reduction of absorbers. Finally, the number of absorbers was calculated by multiplying the density by the sample volume of 0.445 mm3. The average number density and number of absorbers of all twelve repetitions of collagen solution experiments for each condition is presented in Table 5-5 with their corresponding standard deviations with respect to the cumulative num ber of photons. The number densities have been plotted for each perturbation energy in Figures 5-11, 5-12, 5-13 and 5-14 for collagen solutions. Linear regression curve fits may be applied to determine the reduction in number density per photon for each condition studied. The resulting curve fits are displayed on the plots and the R2 values are all greater than 0. 979. These slopes have been plotte d verses perturbation energy in Figure 5-15. These slopes can easily be used to determine the number of photons required to break a single peptide bond by multiplying by the sample volume and taking the inverse. These results are presented in Table 5-6. In both the plot and the table, the values are consistent with each other, particularly for the last three en ergies. The lowest energy of 0.55 mJ/pulse was obtained by reducing the output of the GAM laser, and as the laser output is reduced, the less reliable the output energy become s. Including the 0.55 mJ/pulse da ta point, the average number of photons to break a peptide bond is 28. Exclud ing the 0.55 mJ/pulse data point, the average number of photons to br eak a peptide bond is 29. Lastly, the synchronized collagen data is anal yzed to find the contribution of the peptide bond to the transient reduction of extinction coe fficient. The synchronize d extinction coefficient may be defined by the following equations. AA ext P ext dn n sync extK K K (5-10) n dn N d N n dn N d N KAA AA n AA AA P P n P P dn n sync ext ) ( ) (, (5-11)

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109 This equation can be solved for dn N dP P) ( and simplified into the following equation: n n dn K d N N K K dn K d dn N dAA o AA o Coll dn n unsync ext dn n sync ext P P P) ( ) ( ) (, (5-12) The term dn K dAA) ( is constant and was calculated us ing the amino acid experiments above. sync extK, and unsync extK,have been calculated for each time st ep for each perturbation energy. The value n is the number of photons per pulse. Due to the different concentrations of the amino acid and collagen solutions, a correction factor was placed in front of the dn K dAA) (term to compensate. This factor is the ratio of the init ial overall number density of the collagen solution to the initial overall number de nsity of the amino aci d solution. In this manner, the dynamic change in extinction coefficient due to the peptide bonds per photon was determined for each time step. The values of dn K dP) (are tabulated in Table 5-7. A dditionally, these values were normalized by the number of peptide bonds. The calculated value of dn K dAA) ( is -1.24E-14, =2.43E-15) and its normalized value is -1.09E-34, =2.29E-35 as determined by dividing by the number of amino acids, can be compared with these values. The change in the dynamic extinction coefficient (synchronized) per photon has two contributions: amino acids and peptide bonds. By comparing the normalized values, it is not ed that on average, the portion due to amino acids is only 5% of the por tion due to peptide bonds.

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110 In an attempt to see any changes in th e trends of synchroni zed and unsynchronized transmission during the laser pulse timing, the ra tio of the transmitted to the incident peak was calculated for each time step in the laser pulse. For this exercise, 10 re corded spectra were averaged at 0.77 mJ/pulse for the synchronized and unsynchronized cases. The ratios were taken and plotted in Figure 5-16. The s ynchronized transmission ratio is always slightly greater than the unsynchronized ratio as expected; however, th ere is no clear difference in the transmitted ratio trends between the synchr onized and unsynchronized cases. With regards to the dynamic component, for both collagen and amino acid solutions, the measured number of absorbers in the sample is less in the synchronized case than in the unsynchronized case. The observation indicates transient reduction in number of absorbers in the sample that is present on the order of nanosec onds following perturbation, but has resolved on the order of tens of seconds. In the case of amino acid solutions, th e dynamic reduction is the only reduction that occurs, while the steady state number of ab sorbers remains unchanged. In stark contrast, the collagen so lutions show both a dynamic and a long-term reduction in the number of absorbers. The source of the dynami c reduction is most likel y photobleaching (which is not intended to imply saturation) of the sample, wherein the sample becomes temporarily excited by the incident photons. The excited state has a reduced (or zero) cross-section. This excitation relaxes over time leaving no permanent ch emical change in the solution. Recalling that the difference between the collag en and amino acid solutions is the presence of peptide bonds, the permanent change induced in the collagen sa mples must be due to scission of the peptide bonds in the solution. This confirms that the peptide bond is the primary chromophore in the case of sub-ablative perturbations in th e range of 0.55 to 1.2 mJ/pulse.

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111 355-nm Perturbations An example of the raw incident and transmitted signal spectra obtained from the oscilloscope is shown in Figure 5-17. In this figure, only the probe peaks are of interest. The detectors pick up some small amount of refl ections of the non-delayed 193-nm light. One detector was temporarily relocated in order to measure the time difference between the 355-nm perturbation and 193-nm probe beams, as shown in Figure 5-18. In the figure, the peak-to-peak pulse separation is 19 ns. The average pulse-topulse separation was calculated as 19 ns. Three timing measurements were taken before and after each film was analyzed. As this separation is created with a delay generator, there is jitter in the time separation resulting from the internal jitter of both the excimer and the Nd:YAG lasers. This jitter is approximately +/3 ns. In order to convert this data into transmission, the transmitt ed and incident probe beam signals were first summed, then the background signal was subtracted from the sums, and the ratio of the two was taken. It is again noted that the background (acetic acid) signal did not vary with number of shots applied. Amino acid solutions were not ex amined with the 355-nm perturbation source, as the photon energy is less than in the case of the 194-nm pertur bations. As no long-term changes were noted with amino acid solu tions with the more energetic perturbation source, none are anticipated with this less energetic perturbation source. Using Equation 5-4, the absorbance for each collagen sample was calculated for each time increment and pulse energy for both timing sc hemes (synchronized and unsynchronized). The average absorbance values of all twelve repetiti ons for each condition are presented in Table 5-8 with their corresponding standard deviations. The extinction coefficient (N ) was next calculated by dividing the absorbance by the pathlength of 0.0084 cm. The extinction coeffici ent has been tabulated and plotted for both

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112 timing variations and all energies with respect to the number of incident photons as calculated using Equations 5-5 and 5-6 (Tab le 5-9, Figures 5-19 and 5-20). The extinction coefficient has no slope in the 0.48 mJ/pulse case. In the case of 1.2 mJ/pulse pert urbations, there is a slight downward trend, but not a signifi cant one relative to the 193-n m perturbations. The number of photons delivered in this case is nearly twice the maximum de livered for the case of 193-nm perturbations (1.05 mJ/pulse). There is a change of less than only 2.4 cm-1 with 5.24E16 incident photons compared to 51.3 cm-1 with 4.49E16 incident photons. This corresponds to 508 incident photons required to break a bond. This is extinction coefficient reduction is only 4.5% of that observed for 1.05 mJ/pulse. The collagen solutions for 355-m perturbations also show both a dynamic and a long-term reduction in the number of absorbers, likely due to photobleaching (which is not intended to imply saturation) of the sample, wherein the samp le becomes temporarily excited by the incident photons. The excited state has a re duced (or zero) cro ss-section. The 355-nm permanent change induced in the collagen samples is very sm all compared to the ch anges induced by 193-nm perturbations. This change may be due to scissi on of the peptide bonds in the solution; however, relative to 193-nm perturbation, very few peptide bonds are broken. This is an anticipated result, as at longer wavelengths, the mechanism of ablati on is suggested as thermal in nature. In an extensive review, Vogel and Venugopalan conclu ded that ablation at 193-nm includes both photochemical and photothermal processes, with photochemical component s taking a significant role. Particularly, photochemical processes be come dominant around wavelengths less than 200 nm, with thermal processes more relevant at greater wavelengths. Thus, the 355-nm perturbations results also confirm that the pe ptide bond is the primary chromophore in the case of sub-ablative 193-nm perturbations in the ra nge of 0.55 to 1.2 mJ/pulse (9 to 21 mJ/cm2).

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113 Sub-Ablative Summary The sub-ablative 193-nm and 355-nm perturbati ons yielded insight into 193-nm and 355nm excitation of collagen and amino acids. Ami no acid solutions were not permanently altered by 193-nm sub-ablative energies. For collagen so lutions, an average of 28 photons at 193 nm was required to break a single pe ptide bond. In contrast, perturba tions of collagen solutions by 355-nm resulted in only 4.5% of the extinction coefficient obtained by 193-nm perturbations. Additionally, 508 incident photons at 355-nm were required to break a bond. A dynamic photobleaching (which is not intended to imply saturation) occurs at both 193nm and 355-nm perturbations, wherein the sample becomes temporarily excited by the incident photons. The excited state has a re duced (or zero) crosssection. This effect is approximately 338% greater at 193 nm than at 355 nm. It is also present for both amino acid and collagen solutions. The effect resolves itself by a time s cale of some seconds and does not have a longterm affect on the solutions. The source of th is dynamic reduction is most likely photobleaching of the sample, wherein the sample becomes tem porarily excited by the in cident photons to an electronic state with a re duced absorption coefficient. This excitation relaxes over time leaving no permanent chemical change in the solution. R ecalling that the differen ce between the collagen and amino acid solutions is the presence of pept ide bonds, the permanent change induced in the collagen samples must be due to sc ission of the peptide bonds in th e solution. This confirms that the peptide bond is the primary chromophore in the case of subablative perturbations in the range of 0.55 to 1.2 mJ/pulse (9 to 21 mJ/cm2). These results point to a photochemical mechanism of sub-ablative perturbations of collage n solution. It was previously supposed that at sub-ablative conditions only thermal mechanisms were present. This study contradicts this belief, as photothermal mechanisms are quantified.

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114 Figure 5-1. Schematic of the sub-ablative experimental setup us ing 193-nm perturbation Figure 5-2. Schematic of the sub-ablative experimental setup us ing 355-nm perturbation 193-nm Line Filter Optical Density Fil te r s 193-nm ArF Mirror 193-nm ArF Glass Quartz Pin Hole Pin Hole Pin Hole Mirro r Mirror Mirro r Detector Beam Dump Beam Dump Sample Cell Oscilloscope 193-nm ArF 355-nm Nd:YAG Optical Density Filters 193-nm Line Filter Detector Incident Signal Transmitted Signal 193-nm Line Filter Optical Density Filt e r s 193-nm ArF Mirror 193-nm ArF Glass Quartz Pin Hole Pin Hole Pin Hole 193-nm Line Filter Optical Density Filters Detector Mirro r Mirror Mirro r Detector Mirro r Beam Dump Beam Dump Sample Cell Oscilloscope Transmitted Signal Incident Signal

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115 Figure 5-3. Schematic of the sample cell us ed in the sub-ablative experimental setup -0.005 0 0.005 0.01 0.015 0.02 -3-2-10123Signal (arb. units)Beam Radius (mm) Figure 5-4. Representative profile of the 193-nm perturbation beam O-Ring Quartz Flat Sample Solution Quartz Flat Perturbing Pulse Probing Pulse Probe Beam 1.8-mm Dia.

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116 -0.005 0 0.005 0.01 0.015 0.02 0.025 0.03 -60-40-200204060 Incident TransmittedSignal (arb. units)Time (ns) Figure 5-5. Example of oscillosc ope output for 193-nm perturbation Probe Beam Signals Perturbation Beam Signals

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117 Table 5-1. Average absorbance values and stan dard deviations for each pulse energy and time step with 193-nm perturba tion of collagen solutions Perturbation Number Synchronized Synchronized Un Synchronized Un Synchronized energy (mJ/pulse) of pulses Average absorbance Standard deviation absorbance Average absorbance Standard deviation absorbance 0.55 0 0.746 0.084 0.746 0.084 11 0.602 0.056 0.700 0.058 22 0.579 0.057 0.669 0.060 33 0.563 0.049 0.648 0.049 44 0.541 0.054 0.621 0.051 55 0.517 0.042 0.610 0.047 66 0.503 0.050 0.578 0.051 77 0.485 0.041 0.561 0.048 88 0.469 0.048 0.545 0.049 99 0.446 0.039 0.527 0.044 0.77 0 0.877 0.035 0.877 0.035 11 0.733 0.027 0.854 0.031 22 0.708 0.032 0.821 0.028 33 0.669 0.026 0.779 0.033 44 0.647 0.023 0.752 0.027 55 0.623 0.024 0.732 0.030 66 0.599 0.028 0.708 0.026 77 0.584 0.024 0.680 0.027 88 0.559 0.021 0.665 0.028 99 0.542 0.023 0.641 0.023 0.93 0 0.849 0.082 0.849 0.082 11 0.699 0.065 0.821 0.073 22 0.663 0.069 0.776 0.082 33 0.620 0.053 0.730 0.074 44 0.590 0.056 0.695 0.071 55 0.560 0.044 0.665 0.068 66 0.525 0.060 0.623 0.085 77 0.500 0.053 0.602 0.077 88 0.484 0.060 0.572 0.077 99 0.447 0.062 0.547 0.078 1.05 0 0.884 0.044 0.884 0.044 11 0.684 0.039 0.844 0.041 22 0.645 0.036 0.792 0.041 33 0.611 0.037 0.756 0.042 44 0.574 0.038 0.720 0.039 55 0.547 0.039 0.686 0.035 66 0.522 0.036 0.653 0.034 77 0.499 0.035 0.622 0.031 88 0.472 0.033 0.578 0.026 99 0.453 0.040 0.570 0.027

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118 Table 5-2. Average absorbance values and stan dard deviations for each pulse energy and time step with 193-nm perturba tion of amino acid solutions Perturbation Number Synchronized Synchronized Un Synchronized Un Synchronized energy (mJ/pulse) of pulses Average absorbance Standard deviation absorbance Average absorbance Standard deviation absorbance 0.93 0 0.453 0.034 0.453 0.034 11 0.355 0.033 0.451 0.037 22 0.364 0.035 0.458 0.038 33 0.363 0.035 0.461 0.041 44 0.364 0.035 0.461 0.044 55 0.365 0.037 0.460 0.043 66 0.368 0.035 0.459 0.040 77 0.366 0.038 0.459 0.045 88 0.372 0.038 0.464 0.042 99 0.371 0.039 0.461 0.044

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119 Table 5-3. Average extinction coefficient (cm-1) values and standard deviations for each pulse energy and time step with 193-nm perturbation of collagen solutions Perturbation Cumulative Synchronized Synchronized Un Synchronized Un Synchronized energy (mJ/pulse) photons Average extinction coefficient (cm 1) Standard deviation extinction coefficient (cm 1) Average extinction coefficient (cm 1) Standard deviation extinction coefficient (cm 1) 0.55 0.00E+00 89.00 10.01 89.00 10.01 2.61E+15 71.85 6.66 83.54 6.93 5.22E+15 69.09 6.79 79.84 7.20 7.83E+15 67.12 5.85 77.31 5.90 1.04E+16 64.56 6.39 74.05 6.09 1.31E+16 61.71 5.01 72.83 5.63 1.57E+16 59.95 5.99 69.02 6.07 1.83E+16 57.91 4.95 66.98 5.68 2.09E+16 56.00 5.75 65.01 5.83 2.35E+16 53.19 4.68 62.83 5.22 0.77 0.00E+00 104.68 4.20 104.68 4.20 3.65E+15 87.45 3.22 101.92 3.67 7.31E+15 84.45 3.88 97.95 3.37 1.10E+16 79.87 3.15 92.95 3.89 1.46E+16 77.21 2.69 89.73 3.19 1.83E+16 74.32 2.90 87.32 3.55 2.19E+16 71.51 3.35 84.47 3.06 2.56E+16 69.69 2.91 81.10 3.17 2.92E+16 66.73 2.55 79.33 3.32 3.29E+16 64.61 2.72 76.43 2.74 0.93 0.00E+00 101.27 9.81 101.27 9.81 4.41E+15 83.35 7.72 97.91 8.74 8.83E+15 79.12 8.21 92.62 9.73 1.32E+16 73.94 6.34 87.14 8.87 1.77E+16 70.40 6.63 82.97 8.43 2.21E+16 66.86 5.25 79.37 8.09 2.65E+16 62.59 7.16 74.31 10.13 3.09E+16 59.68 6.30 71.78 9.20 3.53E+16 57.68 7.11 68.25 9.20 3.97E+16 53.37 7.43 65.22 9.30 1.05 0.00E+00 105.41 5.21 105.41 5.21 4.98E+15 81.64 4.67 100.68 4.85 9.97E+15 77.00 4.26 94.44 4.92 1.50E+16 72.92 4.46 90.16 4.95 1.99E+16 68.47 4.56 85.86 4.64 2.49E+16 65.22 4.66 81.86 4.13 2.99E+16 62.29 4.28 77.88 4.02 3.49E+16 59.51 4.17 74.26 3.69 3.99E+16 56.34 3.94 68.94 3.16 4.49E+16 54.08 4.80 68.01 3.17

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120 Table 5-4. Average extinction coefficient (cm-1) values and standard deviations for each pulse energy and time step with 193-nm pe rturbation of amino acid solutions Perturbation Cumulative Synchronized Synchronized Un Synchronized Un Synchronized energy (mJ/pulse) photons Average extinction coefficient (cm 1) Standard deviation extinction coefficient (cm 1) Average extinction coefficient (cm 1) Standard deviation extinction coefficient (cm 1) 0.93 0.00E+00 54.07 4.12 54.07 4.12 4.41E+15 42.37 3.90 53.78 4.41 8.83E+15 43.37 4.15 54.68 4.55 1.32E+16 43.30 4.21 55.02 4.94 1.77E+16 43.48 4.22 55.00 5.22 2.21E+16 43.58 4.46 54.91 5.16 2.65E+16 43.88 4.22 54.80 4.78 3.09E+16 43.62 4.48 54.79 5.36 3.53E+16 44.34 4.59 55.39 4.99 3.97E+16 44.25 4.65 54.97 5.21 40 50 60 70 80 90 100 -5 101505 10151 10161.5 10162 10162.5 1016 0.55 mJ/pulse Synchronized 0.55 mJ/pulse UnsynchronizedExtinction Coefficient (cm-1)Photons Figure 5-6. Average extinction coefficient ( N) and standard deviat ions for 0.55 mJ/pulse energy and time step with 193-nm perturbation of collagen solutions

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121 40 50 60 70 80 90 100 110 -5 101505 10151 10161.5 10162 10162.5 10163 10163.5 1016 0.77 mJ/pulse Synchronized 0.77 mJ/pulse UnsynchronizedExtinction Coefficient (cm-1)Photons Figure 5-7. Average extinction coefficient ( N) and standard deviat ions for 0.77 mJ/pulse energy and time step with 193-nm perturbation of collagen solutions 40 50 60 70 80 90 100 110 120 -1 101601 10162 10163 10164 1016 0.93 mJ/pulse Synchronized 0.93 mJ/pulse UnsynchronizedExtinction Coefficient (cm-1)Photons Figure 5-8. Average extinction coefficient ( N) and standard deviat ions for 0.93 mJ/pulse energy and time step with 193-nm perturbation of collagen solutions

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122 40 50 60 70 80 90 100 110 120 -1 101601 10162 10163 10164 10165 1016 1.05 mJ/pulse Synchronized 1.05 mJ/pulse UnsynchronizedExtinction Coefficient (cm-1)Photons Figure 5-9. Average extinction coefficient ( N) and standard deviat ions for 1.05 mJ/pulse energy and time step with 193-nm perturbation of collagen solutions 30 35 40 45 50 55 60 65 70 -1 101601 10162 10163 10164 1016 0.93 mJ/pulse Synchronized 0.93 mJ/pulse UnsynchronizedExtinction Coefficient (cm-1)Photons Figure 5-10. Average extinction coefficient ( N) and standard deviat ions for 0.93 mJ/pulse energy and time step with 193-nm pe rturbation of amino acid solutions

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123 Table 5-5. Average bond number densities (peptide bonds/ml) and numbers of absorbers (peptide bonds) and standard deviations for each pulse energy and time step with 193nm perturbation of collagen solutions Perturbation Cumulative Peptide Peptide Peptide Peptide energy (mJ/pulse) photons number density (bonds/ml) number density standard deviation (bonds/ml) number density (bonds) number density standard deviation (bonds) 0.55 0.00E+00 7.48E+18 8.41E+17 3.33E+15 3.74E+14 2.61E+15 6.96E+18 7.01E+17 3.10E+15 3.12E+14 5.22E+15 6.63E+18 6.76E+17 2.95E+15 3.01E+14 7.83E+15 6.41E+18 5.75E+17 2.85E+15 2.56E+14 1.04E+16 6.13E+18 5.72E+17 2.73E+15 2.55E+14 1.31E+16 6.02E+18 5.37E+17 2.68E+15 2.39E+14 1.57E+16 5.69E+18 5.67E+17 2.53E+15 2.52E+14 1.83E+16 5.51E+18 5.41E+17 2.45E+15 2.41E+14 2.09E+16 5.34E+18 5.47E+17 2.37E+15 2.44E+14 2.35E+16 5.15E+18 5.00E+17 2.29E+15 2.23E+14 0.77 0.00E+00 8.80E+18 3.53E+17 3.91E+15 1.57E+14 3.65E+15 8.57E+18 3.53E+17 3.81E+15 1.57E+14 7.31E+15 8.22E+18 3.22E+17 3.66E+15 1.43E+14 1.10E+16 7.78E+18 3.61E+17 3.46E+15 1.61E+14 1.46E+16 7.50E+18 3.11E+17 3.34E+15 1.38E+14 1.83E+16 7.29E+18 3.31E+17 3.24E+15 1.47E+14 2.19E+16 7.04E+18 2.96E+17 3.13E+15 1.32E+14 2.56E+16 6.74E+18 2.97E+17 3.00E+15 1.32E+14 2.92E+16 6.59E+18 3.11E+17 2.93E+15 1.39E+14 3.29E+16 6.34E+18 2.67E+17 2.82E+15 1.19E+14 0.93 0.00E+00 8.51E+18 8.24E+17 3.79E+15 3.67E+14 4.41E+15 8.22E+18 8.39E+17 3.66E+15 3.73E+14 8.83E+15 7.75E+18 9.05E+17 3.45E+15 4.03E+14 1.32E+16 7.27E+18 8.48E+17 3.24E+15 3.77E+14 1.77E+16 6.91E+18 8.01E+17 3.07E+15 3.56E+14 2.21E+16 6.59E+18 7.68E+17 2.93E+15 3.42E+14 2.65E+16 6.15E+18 9.31E+17 2.74E+15 4.14E+14 3.09E+16 5.93E+18 8.80E+17 2.64E+15 3.92E+14 3.53E+16 5.62E+18 8.67E+17 2.50E+15 3.86E+14 3.97E+16 5.35E+18 8.75E+17 2.38E+15 3.90E+14 1.05 0.00E+00 8.86E+18 4.38E+17 3.94E+15 1.95E+14 4.98E+15 8.46E+18 4.62E+17 3.76E+15 2.06E+14 9.97E+15 7.91E+18 4.63E+17 3.52E+15 2.06E+14 1.50E+16 7.54E+18 4.67E+17 3.35E+15 2.08E+14 1.99E+16 7.16E+18 4.42E+17 3.19E+15 1.97E+14 2.49E+16 6.81E+18 3.96E+17 3.03E+15 1.76E+14 2.99E+16 6.46E+18 3.81E+17 2.88E+15 1.69E+14 3.49E+16 6.15E+18 3.53E+17 2.74E+15 1.57E+14 3.99E+16 5.68E+18 3.05E+17 2.53E+15 1.36E+14 4.49E+16 5.60E+18 2.98E+17 2.49E+15 1.33E+14

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124 4 10185 10186 10187 10188 10189 1018-5 101505 10151 10161.5 10162 10162.5 1016 y = 7.2576e+18 92.516x R2= 0.97963 Number Density (bonds/ml)Photons Figure 5-11. Average number de nsities (bonds/ml) of peptide bonds and standard deviations for 0.55 mJ/pulse energy and time step with 193nm perturbation of collagen solutions 6 10186.5 10187 10187.5 10188 10188.5 10189 10189.5 1018-5 101505 10151 10161.5 10162 10162.5 10163 10163.5 1016 y = 8.7295e+18 76.216x R2= 0.9894 Number Density (bonds/ml)Photons Figure 5-12. Average number densities (bonds/ml) of peptide bonds and standard deviations for 0.77 mJ/pulse energy and time step with 193nm perturbation of collagen solutions

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125 4 10185 10186 10187 10188 10189 10181 1019-1 101601 10162 10163 10164 1016 y = 8.451e+18 81.606x R2= 0.99214 Number Density (bonds/ml)Photons Figure 5-13. Average number densities (bonds/ml) of peptide bonds and standard deviations for 0.93 mJ/pulse energy and time step with 193nm perturbation of collagen solutions 4 10185 10186 10187 10188 10189 10181 1019-1 101601 10162 10163 10164 10165 1016 y = 8.7217e+18 74.514x R2= 0.99109 Number Density (bonds/ml)Photons Figure 5-14. Average number de nsities (bonds/ml) of peptide bonds and standard deviations for 1.05 mJ/pulse energy and time step with 193nm perturbation of collagen solutions

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126 0 20 40 60 80 100 0.50.60.70.80.911.1Number Density/PhotonPerturbation Energy (mJ/pulse) Figure 5-15. Number density change per photon for each pulse energy with 193-nm perturbation of collagen solutions Table 5-6. Number of photons required to break a peptide bond for each pulse energy with 193nm perturbation of collagen solutions Perturbation Photons per broken Energy (mJ/pulse) peptide bond 0.55 24.29 0.77 29.48 0.93 27.54 1.05 30.16

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127 Table 5-7. Average number de nsities (peptide bonds/ml) and numbers of absorbers (peptide bonds) and standard deviations for each pul se energy and time step with 193-nm perturbation of collagen solutions Perturbation energy (mJ/pulse) Cumulative photons dn K dP) ( ( cm 1 per photon) dn K dP) ( stand. dev. ( cm 1 per photon) Normalized dn K d NP Coll) ( 1 (cm 1 per photon per peptide bond) Normalized dn K d NP Coll) ( 1 stand. dev. ( cm 1 per photon per peptide bond) 0.55 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 2.61E+15 2.11E 14 1.80E 14 3.01E 33 2.59E 33 5.22E+15 1.93E 14 1.85E 14 2.89E 33 2.79E 33 7.83E+15 1.83E 14 1.56E 14 2.83E 33 2.43E 33 1.04E+16 1.70E 14 1.65E 14 2.75E 33 2.70E 33 1.31E+16 2.00E 14 1.41E 14 3.30E 33 2.35E 33 1.57E+16 1.62E 14 1.60E 14 2.82E 33 2.81E 33 1.83E+16 1.62E 14 1.41E 14 2.91E 33 2.56E 33 2.09E+16 1.61E 14 1.53E 14 2.99E 33 2.87E 33 2.35E+16 1.72E 14 1.31E 14 3.32E 33 2.56E 33 0.77 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 3.65E+15 1.84E 14 6.53E 15 2.15E 33 7.68E 34 7.31E+15 1.71E 14 6.87E 15 2.08E 33 8.41E 34 1.10E+16 1.65E 14 6.70E 15 2.13E 33 8.68E 34 1.46E+16 1.58E 14 5.58E 15 2.11E 33 7.51E 34 1.83E+16 1.64E 14 6.14E 15 2.26E 33 8.50E 34 2.19E+16 1.64E 14 6.07E 15 2.33E 33 8.69E 34 2.56E+16 1.43E 14 5.76E 15 2.12E 33 8.61E 34 2.92E+16 1.59E 14 5.60E 15 2.42E 33 8.60E 34 3.29E+16 1.48E 14 5.17E 15 2.35E 33 8.24E 34 0.93 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 4.41E+15 1.52E 14 1.29E 14 1.85E 33 1.58E 33 8.83E+15 1.40E 14 1.41E 14 1.81E 33 1.83E 33 1.32E+16 1.37E 14 1.21E 14 1.88E 33 1.67E 33 1.77E+16 1.30E 14 1.19E 14 1.88E 33 1.73E 33 2.21E+16 1.29E 14 1.07E 14 1.96E 33 1.64E 33 2.65E+16 1.20E 14 1.37E 14 1.96E 33 2.25E 33 3.09E+16 1.25E 14 1.24E 14 2.10E 33 2.11E 33 3.53E+16 1.08E 14 1.29E 14 1.92E 33 2.31E 33 3.97E+16 1.22E 14 1.32E 14 2.28E 33 2.49E 33 1.05 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 4.98E+15 1.77E 14 6.61E 15 2.10E 33 7.91E 34 9.97E+15 1.61E 14 6.38E 15 2.04E 33 8.17E 34 1.50E+16 1.59E 14 6.54E 15 2.12E 33 8.79E 34 1.99E+16 1.61E 14 6.38E 15 2.25E 33 9.04E 34 2.49E+16 1.54E 14 6.10E 15 2.26E 33 9.08E 34 2.99E+16 1.43E 14 5.76E 15 2.22E 33 9.04E 34 3.49E+16 1.35E 14 5.46E 15 2.20E 33 9.01E 34 3.99E+16 1.14E 14 4.96E 15 2.01E 33 8.83E 34 4.49E+16 1.27E 14 5.64E 15 2.27E 33 1.02E 33

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128 0.2 0.25 0.3 0.35 0.4 02468101214 Unsynchronized SynchronizedTransmitted RatioTime (ns) Figure 5-16. Transmission ratio with respect to time during laser pulse -0.005 0 0.005 0.01 0.015 0.02 0.025 -60-40-200204060 Incident TransmittedSignal (arb. units)Time (ns) Figure 5-17. Example of oscillosc ope output for 355-nm perturbation

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129 0 0.002 0.004 0.006 0.008 0.01 -60-40-200204060Signal (arb. units)Time (ns) Figure 5-18. Average delay for 355-nm perturbation Table 5-8. Average absorbance values and stan dard deviations for each pulse energy and time step with 355-nm perturba tion of collagen solutions Perturbation Number Synchronized Synchronized Un Synchronized Un Synchronized Energy (mJ/pulse) of pulses Average absorbance Standard deviation absorbance Average absorbance Standard deviation absorbance 0.48 0 1.033 0.033 1.033 0.033 11 0.999 0.037 1.042 0.031 22 0.980 0.033 1.031 0.047 33 0.985 0.045 1.035 0.045 44 1.003 0.041 1.017 0.045 55 0.976 0.042 1.033 0.029 66 0.973 0.046 1.033 0.043 77 0.976 0.040 1.031 0.042 88 0.977 0.046 1.041 0.062 99 0.954 0.049 1.012 0.033 1.2 0 0.694 0.030 0.694 0.030 11 0.686 0.036 0.698 0.034 22 0.686 0.032 0.692 0.034 33 0.677 0.033 0.692 0.034 44 0.676 0.035 0.692 0.036 55 0.674 0.038 0.696 0.037 66 0.669 0.037 0.676 0.035 77 0.660 0.040 0.670 0.037 88 0.660 0.034 0.676 0.035 99 0.647 0.040 0.664 0.035

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130 Table 5-9. Average extinction coefficients a nd standard deviations for each pulse energy and time step with 355-nm perturbation of collagen solutions Perturbation Cumulative Synchronized Synchronized Un Synchronized Un Synchronized energy (mJ/pulse) photons Average extinction coefficient (cm 1) Standard deviation extinction coefficient (cm 1) Average extinction coefficient (cm 1) Standard deviation extinction coefficient (cm 1) 0.48 0.00E+00 123.245 3.988 123.245 3.988 4.19E+15 119.172 4.443 124.330 3.749 8.38E+15 116.868 3.945 122.985 5.649 1.26E+16 117.528 5.320 123.443 5.322 1.68E+16 119.709 4.934 121.320 5.399 2.10E+16 116.426 5.016 123.213 3.483 2.51E+16 116.043 5.449 123.225 5.165 2.93E+16 116.399 4.794 123.056 5.005 3.35E+16 116.549 5.491 124.231 7.423 3.77E+16 113.769 5.848 120.754 3.879 1.2 0.00E+00 82.746 3.582 82.746 3.582 1.05E+16 81.844 4.349 83.238 4.058 2.10E+16 81.863 3.813 82.560 4.037 3.14E+16 80.805 3.969 82.582 4.068 4.19E+16 80.622 4.208 82.503 4.311 5.24E+16 80.419 4.573 82.980 4.365 6.29E+16 79.760 4.443 80.602 4.154 7.33E+16 78.719 4.796 79.959 4.362 8.38E+16 78.708 4.100 80.630 4.192 9.43E+16 77.193 4.776 79.186 4.205

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131 100 105 110 115 120 125 130 135 140 -1 101601 10162 10163 10164 1016 0.48 mJ/pulse Synchronized 0.48 mJ/pulse UnsynchronizedExtinction Coefficient (cm-1)Photons Figure 5-19. Average extinction coefficient ( N) and standard deviations for 0.48mJ/pulse energy and time step with 355-nm perturbation of collagen solutions 70 75 80 85 90 95 -2 101602 10164 10166 10168 10161 1017 1.2 mJ/pulse Synchronized 1.2 mJ/pulse UnsynchronizedExtinction Coefficient (cm-1)Photons Figure 5-20. Average extinction coefficient ( N) and standard deviations for 1.2 mJ/pulse energy and time step with 355-nm perturbation of collagen solutions

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132 CHAPTER 6 MASS SPECTROSCOPY Mass spectrometry of the ablati on plumes is an area of resear ch in photoablation of corneas that is ripe for re-evaluation. Better technology, including an advan ced surgical lase r with a clean energy distribution and a new ma ss spectrometry system shall a llow for a better experimental setup. Knowledge of the plume contents may lead to a better understandin g of the process as a whole, notably in conjunction with th e collagen and amino acid studies. Experimental Setup and Methods Mass spectrometry was perfor med with Hiden Analyticals HPR20-QIC atmospheric gas analysis system. This quadrupole mass spectromete r is compact, and has a sniffer probe that allows direct sampling of the ablation plume fr om the corneal surface at atmospheric pressure. This machine has a sensitivity of about 15 ppb an d a mass detection range of up to 200 amu, and was operated at 3 x 10-5 torr. Bovine eyes were harvested as described in Chapter 2 above, and the same delivery system was used to generate ab lations of the corneas, as shown in Figure 6-1. The probe of the portable mass spectrometer was mounted approximately 2 mm above the corneal surface at the edge of th e 6-mm ablation zone. Treatments of 9 diopters at 100 Hz were applied to the bovine corneas wh ile the mass spectrometer probe sampled the air, bringing the sample to the mass spectrometer using a heated quartz capillary tube. Two ablations were applied for each cornea. For a range of 1 to 100 amu, 7 scans from were recorded with 0.25-amu resolution for each of 12 ablations (6 eyes). For a range of 101 to 150 amu, 7 scans were recorded with 0.25-resolution for each of 4 ab lations (2 eyes). Bac kground scans were also recorded from the same location in order to determine the baseline contribution from the local environment. Background spectra were recorded w ith the laser operating in order to include any

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133 effects of laser-air inte raction, such as ozone formation. A set of 7 background scans was taken for each ablation. Mass Spectrometry Results The background scans are presented first. The average background for all scans and all eyes over the entire mass range ( 1 amu) studied is shown in Fi gure 6-2. It is clear from the plot that the components of th e environmental air are below 45 amu. Thusly, Figure 6-3 focuses on that mass range. In this figure, all peaks have been labeled with thei r corresponding molecular component. Any additional compon ents relative to this backgr ound signal may indicate ablation products. Just as with the background scans, the signals obtained during corneal ablations from all scans and all eyes were averaged together. The results are shown in Figure 6-4. There are three peaks of interest in this fi gure, at 17, 18 and 42 amu. The peaks at 17 and 18 are OH and H2O, respectively; their increase is due to water released as an ablation product. This is consistent with previous results which indicate the presence of water vapor in the ablation products [68]. The third peak at 42 amu, as shown in Figure 6-5, is unique to the ablation spectra; therefore, a candidate to originate fr om an ablation product. An analysis of the collagen structure (Figure 6-6) shows one possibility for the origin of the 42-amu component: a fragment of glycine containing 2 carbons, 2 hydrogens and 1 oxygen unit. This unit is depicted in Figure 6-7. As the peptide bond is the expected primary chromophore, a NH grouping at 29 amu is expected to be present with this fragment. An additional contribution to the 29 am u signal is not present in the ablation series as compared to the background series. However, the expected c ontribution due to a NH ablation product is very small relative to the contribut ion from the surrounding air.

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134 Mass Spectrometry Summary Mass spectroscopy of ablation plumes has show n a large amount of water present in the ablation products, as well as a possible 42-amu fragment of glycine containing 2 carbon units, 2 hydrogen units and one oxygen unit. This data is consistent with the findings of Hahn et al in 2004, which found that water vapor is the predom inant constituent in the ablation plume. The 42amu fragment is consistent w ith peptide bonds being the primary chromophore, as the glycineproline peptide bond must be broken in the creatio n of this fragment. Th is fragment was not found in previous mass spectroscopic analys es of corneal photoa blation [74, 75].

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135 Figure 6-1. Experimental setup for mass spectrometry Eye Holder Mirror 1 Mirror 2 Lens 2 Scan Cube 193-nm ArF Lens 1 Homogenizer Pin Hole Laser 1 Portable Mass Spectrometer Sniffer Probe

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136 10-910-810-710-610-5020406080100120140160Magnitude (arb. units)Atomic Mass Unit (amu) Figure 6-2. Average background spec tra over entire analyzed range

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137 10-910-810-710-610-551015202530354045Magnitude (arb. units)Atomic Mass Unit (amu) Figure 6-3. Average background sp ectra over range of activity N O OH H2O N2 O2 O2 Ar CO2 H2O N2

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138 10-910-810-710-610-551015202530354045 Ablation Spectra Background SpectraMagnitude (arb. units)Atomic Mass Unit (amu) Figure 6-4. Average bac kground and ablation spectra OH H2O

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139 8 10-99 10-910-82 10-830354045 Ablation Spectra Background SpectraMagnitude (arb. units)Atomic Mass Unit (amu) Figure 6-5. Average background and abla tion spectra focused around 42 amu peak 42

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140 Figure 6-6. Simplified collage n structure of the human co rnea taken from reference 8 Figure 6-7. Structure of the 42-amu molecular fragment C O C H H Broken Peptide Bond Broken C-N Bond C C N C C N C C N O O O C C C (Hydroxyproline) (Glycine) Glycine Proline Hydroxyproline OH C C C

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141 CHAPTER 7 CONCLUSIONS AND FUTURE WORK This chapter presents the important conclusi ons of the preceding chapters and places the results of each experiment in context with each other. Additionally, recommendations for future work are given. Conclusions The current experiments have examined the pot ential effects of laser repetition rate on corneal ablation over a range of clinically relevant conditions Experiments have assessed a comprehensive range of parameters, including plume dynamics, corneal ablation profiles, and high-resolution microscopy of collagen structure, which in aggregate lead to the conclusion that no observable effects of laser re petition rate are present for the comparison of 60 and 400 Hz: The plume dynamics studies (imaging and tr ansmission) indicate that the bulk of the plume evolves away from the corneal surf ace on a time scale of several ms, and is largely gone by 2.5 ms (the pulse-to-pulse time spacing for 400 Hz ablation. Thus attenuation of the laser pulse by the previous plume is ne gligible up to about 400 Hz, as confirmed by the ablation profile study. The ablation rate in bovine corneas wa s calculated to be between 0.95 and 0.98 m/pulse for a total pulse energy of 2.7 mJ and a Gaussian beam profile. The ablation rate of PMMA was cal culated to be approximately 0.18 m/pulse. The ablation threshold, 25 mJ/cm2, was determined using a unique method of comparing the edge of the ablation profile with the beam fluence profile. The ablation rate in bovine corneas was constant for 60 and 400 Hz. The shapes and the depths of the ablation prof iles were likewise consistent. No differential tissue damage was noted in microscopy (visible, SEM, TEM) images of 400 Hz as compared to 60 Hz, including no disorganization, collagen size changes or coagulation. The numbers of fibrils in the TEM images were counted and no statistical difference in collagen fibril de nsity near the ablation surface between 60 and 400 Hz was observed. Additional conclusions may also be drawn from the set of expe riments with regard to the implementation of the laser ablation system:

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142 The addition of a small vacuum (suction) to the corneal surface, which approximated the plume evacuator used by surgeons, di d not alter the abla tion profiles obtained from the excimer system. Reversing the algorithm of the excimer la ser showed no alteration to the ablation profiles achieved on PMMA. While a tre nd was noted of over-ablation by the reversed pattern was seen, this trend wa s dominated by large standard deviations, such that no statistical difference was documented. The sub-ablative 193-nm and 355-nm perturbati ons yielded insight into 193-nm and 355nm excitation of collagen and amino acids. These perturbations are also di scussed in the context of photoablation mechanism. Amino acid solutions were not permanently altered by 193-nm subablative energies. An average of 28 photons at 193 nm was required to break a single peptide bond. Perturbations of collagen solutions by 355-nm resulted in only 4.5% of the extinction coefficient obtained by 193nm perturbations, resulting in 508 bonds being required to break a single peptide bond. A dynamic photobleaching occurs at both 193-nm and 355-nm perturbations. This effect is greater at 193 nm than at 355 nm It is also present for both amino acid and collagen solutions. The effect resolves itsel f by a time scale of tens of seconds and does not have a long-term affect on the solutions. The source of the dynamic reduction is mo st likely photobleachi ng of the sample, wherein the sample becomes temporarily excited by the incident photons to an electronic state with a reduced absorption coefficient. This excitation relaxes over time leaving no permanent chemical change in the solution. Recalling that the difference between the collagen and amino acid solutions is the presence of peptide bonds, the permanent change induced in the collagen samples must be due to scission of the peptide bonds in the solution. This confirms that the peptide bond is the primary chromophore in th e case of sub-ablative perturbations in the range of 0.55 to 1.2 mJ/pulse (9 to 21 mJ/cm2). It was previously supposed that at sub-ab lative conditions only thermal mechanisms were present. This study contradicts this belief, as photothermal mechanisms are definitely existent.

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143 Mass spectrometry experiments were used to analyze the ablation pr oducts present in the ablation plume. The main constituent present wa s found to be water, with a small amount of a 42-amu fragment of possibly glycine contai ning 2 carbon units, 2 hydrogen units and one oxygen unit. In summary, investigation of the relative e ffects of excimer laser repetition rate on the overall corneal ablation processe s (i.e. plume dynamics, ablati on rates and corneal pathology) revealed no measurable difference under conditions typical of refractive procedures. This study suggests that increases in ArF la ser repetition rates for clinical applications (up to ~400 Hz) appear feasible, and therefore justify the pursuit of additional clinical studies. The exact mechanism of photoa blation remains a topic of research [5, 81]. Several interesting findings in the current study are releva nt and provide insight as to the overall process of excimer laser ablation of corneal tissue, namely the TEM analysis and the transmission experiments. First the finding of a persistent diffuse component of the ablation plume as measured by 193-nm absorption, suggests molecula r fragments from the ablation process itself. This finding is supportive of a photochemical me chanism, in which amino acid fragments are created within the tissue matrix and subsequently ejected with the plume. S econdly the lack of any noticeable damage or pertur bation to the collagen fibrils immediately underlying the ablation zone, as seen in the TEM histol ogy experiments, speaks to the pr ecision of excimer laser tissue etching. The lack of any indication of therma l damage in the microscopy images indicates a photochemical process. Together, such results are consistent w ith the photochemical model of ArF laser ablation of corneal stromal tissue, in which the high excimer laser photon energy (6.4 eV) can directly cleave protein strands (i.e. pep tide bonds), forming transient species, in a very dynamic laser-tissue intera ction process [14].

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144 Ishihara and co-workers have studied the surface temperature of cornea during ablation, and found that the peak surface temperature in creases with laser fluence. At 180 mJ/cm2, they reported a value of 240oC surface temperature, and at fluences near 300 mJ/cm2, the temperature is as high as approximately 325oC. These results suggest a ph otothermal component to the ablation mechanism. However, additional measurement by this group determined that the increase in temperature decrea ses rapidly on a time scale of a few hundred microseconds for 193-nm irradiation. This quick dissipation of su rface temperature supports the current findings of no differential thermal damage, as the pulse-pulse time scale is on the order of several ms [66, 82]. Thusly, the thermal energy is expected to diss ipate on a time scale much less than that of the laser repetition rate. Ishihara and co-workers also found that the surface temperature relaxes more quickly for 193-nm irradiation as compared to 248-nm irradiation noting that a thermal component of ablation is considered to increase with increasing wavelength, notably so in the infrared spectral region [82]. Overall, the re duced surface temperature observed with 193-nm irradiation seems to indicate a mechanism that is less reliant on thermal excitation. Additional insight into the pres ent findings may be gained by contrasting the results with a previous study of laser re petition rate effects of CO2 lasers on tissue. The ablation mechanism of CO2 laser-tissue interaction is considered a purely thermal process. For this type of ablation, thermal damage and the resulting ablation rate ha ve been found to signif icantly increase with laser repetition rate [83, 84]. This is in stark contrast to th e results of the current study, which do not indicate any differential tissue damage or a change in ablation depth with repetition rate. This difference may be explained in the context of th ermal relaxation times. This time is on the order of tens of ms for the case of CO2 ablation and on the order of tens of microseconds for ArF ablation. Thus, the critical lase r repetition rate for 193-nm excime r laser ablation is in the range

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145 of tens of kilohertz, much greater than the range examined in the cu rrent study, while the CO2 laser ablation has a crit ical laser repetition ra te of approximately 20 Hz. This supports the findings of no differential thermal tissue damage [84]. Although the exact pa rtitioning between photochemical and photothermal influences re mains unknown, the role of the photothermal component does not have an altering influence as the laser repetition rate is increased to 400 Hz. The current findings are consiste nt with a primarily photochemi cal process with the 193-nm excimer, which is consistent with the literature [5]. Future Work There are several additional experiments that may be performed in order to further shed light on the mechanisms of laser-tissue intera ction. They are briefly summarized in the following: Repeat the mass spectrometry experiments u tilizing a more sensitive instrument in order to uncover further p eaks due to molecular fragments of amino acid chains. Repeat the sub-ablative experiments on bipeptide solutions. Bipeptides are combinations of only two amino acids. Performing these experiments on glycineproline, glycine-hydroxy proline and proline-hydr oxyproline solutions may distinguish which peptide bonds are more likely to break during sub-ablative perturbations. Repeat the sub-ablative experiments with ablative energies. The results could be compared with those found here for sub-abla tive perturbations in order to glean any information on the differences in mechanisms between the two. The experimental setup may need modification, in order to ablate the solution sample and not the sample cell. Repeat the sub-ablative pe rturbation experiments c oncurrent with fluorescence measurements in order to examine fluore scence changes that may occur due to the so-called photobleaching effect. This set of experiments may lead to additional insight into the dynamic response of so lution samples to incident energy. Repeat the sub-ablative experiments with a femtosecond laser. The ultra-short pulses result in nonlinear (multiphoton) abso rption, in contrast to the longer (nanosecond) pulses employed in this work [85]. The use of a femtosecond source would allow the study of the resulting nonlin ear effects.

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146 It has been shown in Chapter 5 that even at low 193-nm fluences, bonds are broken within collagen. At a threshold fluence, the stress buildup due to these broken bonds results in ejection of material from the substrate. Solid mechanics modeling may be performed of the stresses induced by volumetric expansion as a result of broken bonds in order to de termine the critical volu metric stress required for ablation to occur.

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147 APPENDIX A MATLAB EDGE FINDER function find_eye_edge_midpoint %prompt= 'Enter number of standard deviations'; prompt= 'Enter percentage above dark mean' ; numlines=1; defaultanswer= 0.5; standDevs = inputdlg(prompt, 'Input required:' ,1,{num2str(defaultanswer)}); standDevs = str2num(standDevs{1}); saveTo = questdlg ... ( 'Would you like to add new eye data to an existing file?' ); if strcmp(saveTo, 'Yes' ); [saveFile,savePath] = uigetfile( '*.mat' 'Select data file' ); load(fullfile(savePath, saveFile)) fileList = eyeData.fileList; % originalImage = eyeData.originalImage; % croppedImage = eyeData.croppedImage; cropRegion = eyeData.cropRegion; edgeList = eyeData.edgeLocation; elseif strcmp(saveTo, 'No' ) [saveFile, savePath] = uiputfile( '*.mat' 'Save eye edge data to:' ); fileList = cell(0); end if ~exist( 'saveFile' ) | isempty(saveFile) return end theSwitch = 'Yes' ; while strcmp(theSwitch, 'Yes' ) [fileName,pathName] = uigetfile( '*.asc' 'Select file to process' ); if isempty(fileName) break end thisFile = fullfile(pathName, fileName); I = strmatch(thisFile, fileList, 'exact' ); if isempty(fileList) I = 1; elseif ~isempty(I) overWrite = questdlg ... ( 'File has already been processed. Re-do?' 'Yes' 'No' ); if strcmp(overWrite, 'No' ) continue end else I = length(fileList) + 1; end

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148 fileList{I} = thisFile; [thisImage] = dlmread(thisFile, '\t' ); thisImage = thisImage'; rows = 1:size(thisImage,1); cols = 1:size(thisImage,2); % originalImage{I} = thisImage; figure(1) imagesc(cols, rows, thisImage) set(gca, 'ydir' 'normal' ) set(gcf, 'name' [fileName Original' ]) isOK = 'No' ; while strcmp(isOK, 'No' ) figure(1) fprintf( 'Select the crop region.\n' ) p = ginput(2); cutRows = rows(floor(min(p(:,2))):ceil(max(p(:,2)))); cutImage = thisImage(cutRows,:); figure(4) imagesc(cols,cutRows, cutImage) set(gca, 'ydir' 'normal' ) set(gcf, 'name' [fileName Cropped' ]) isOK = questdlg({ 'Is this crop acceptable?' ; ... '(Note: Program needs 50 pixels of eye to average)' }, ... 'Input required:' 'Yes' 'No' 'Yes' ); end % croppedImage{I} = cutImage; cropRegion{I} = cutRows; doneYet = 'No' ; while strcmp(doneYet, 'No' ) for index = 1:size(cutImage,2) theMean = nanmean(cutImage(1:50,index)); theMean2 = nanmean(cutImage(end-50:end,index)); % theSTD = nanstd(cutImage(1:50,index)); % cutOff = theMean + standDevs*theSTD; cutOff = standDevs*(theMean2-theMean)+theMean; overCut = find(cutImage(:,index) > cutOff); diffs = diff(overCut); firstGoods = find(diffs == 1); if firstGoods(2) == (firstGoods(1)+1) edgeLocation(index) = ... mean(cutRows(overCut(firstGoods(1):firstGoods(1)+1))); else edgeLocation(index) = ... mean(cutRows(overCut(firstGoods(2):firstGoods(2)+1))); end end

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149 figure(4) hold on l = plot(cols, edgeLocation, 'k' ); doneYet = questdlg( 'Is this edge acceptable?' 'Input required:' ... 'Yes' 'No' 'Yes' ); if strcmp(doneYet, 'No' ) prompt= 'Enter number of standard deviations' ; numlines=1; defaultanswer= 4; standDevs = inputdlg(prompt, 'Input required:' ,1,{num2str(standDevs)}); standDevs = str2num(standDevs{1}); delete(l) end end edgeList{I} = edgeLocation; eyeData.fileList = fileList; % eyeData.originalImage = originalImage; % eyeData.croppedImage = croppedImage; eyeData.cropRegion = cropRegion; eyeData.edgeLocation = edgeList; save(fullfile(savePath,saveFile), 'eyeData' ) close all theSwitch = questdlg( 'Would you like to do another file?' ... 'Input required:' 'Yes' 'No' 'Yes' ); end save(fullfile(savePath,saveFile), 'eyeData' ) clc

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150 APPENDIX B MATLAB RESULTS COMPILER function compile_results [dataFile, dataPath] = uigetfile( '*.mat' 'Select data file' ); if isempty(dataFile) return end load(fullfile(dataPath, dataFile)) fileList = eyeData.fileList; for index = 1:length(fileList) [junk, thisFile] = fileparts(fileList{index}); thisFile = lower(thisFile); %findEye = findstr(thisFile, 'eye'); findSpace = findstr(thisFile, ); eyeNumber(index) = str2num(thisFile(findSpace(1)+1:findSpace(2)-1)); findPre = findstr(thisFile, 'pre' ); if isempty(findPre) findPost = findstr(thisFile, 'post' ); %runNumber(index) = str2num(thisFile(findPost+4:end));; runNumber = 1; timeNumber(index) = 2; else timeNumber(index) = 1; %runNumber(index) = str2num(thisFile(findPre+3:end)); runNumber = 1; end edgeData(:,index) = eyeData.edgeLocation{index}'; end eyeList = unique(eyeNumber); for eyeIndex = 1:length(eyeList) thisEyePres = find((eyeNumber == eyeList(eyeIndex)) & ... (timeNumber == 1)); preAll = edgeData(:,thisEyePres); fprintf([ 'For eye number num2str(eyeList(eyeIndex)) there are ... num2str(size(preAll,2)) "pre" samples.\n' ]); preMean(:,eyeIndex) = preAll; %preStd(:,eyeIndex) = (std(preAll'))'; thisEyePosts = find((eyeNumber == eyeList(eyeIndex)) & ... (timeNumber == 2)); postAll = edgeData(:,thisEyePosts);

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151 fprintf([ 'For eye number num2str(eyeList(eyeIndex)) there are ... num2str(size(postAll,2)) "post" samples.\n' ]); postMean(:,eyeIndex) = postAll; %postStd(:,eyeIndex) = (std(postAll'))'; end for index = 1:length(eyeList); index pPre(:,index) = (polyfit([1:1024]', preMean(:,index), 2))'; %pPost(:,index) = (polyfit([1:1024]', postMean(:,index), 2))'; fitPre(:,index) = polyval(pPre(:,index), [1:1024]'); %fitPost(:,index) = polyval(pPost(:,index), [1:1024]'); theDiff(:,index) = fitPre(:,index) postMean(:,index); diffP(:,index) = (polyfit([1:1024]', theDiff(:,index), 2))'; diffFit(:,index) = polyval(diffP(:,index), [1:1024]'); rawDiff(:,index) = preMean(:,index) postMean(:,index); SST = sum((preMean(:,index) mean(preMean(:,index))).^2); SSR = sum((fitPre(:,index) mean(preMean(:,index))).^2); RsquarePre(index) = SSR./SST; SST = sum(sum((theDiff(:,index) mean(theDiff(:,index))).^2)); SSR = sum(sum((diffFit(:,index) mean(theDiff(:,index))).^2)); RsquareDiff(index) = SSR./SST; end [dataFile2, dataPath2] = ... uiputfile( '*.txt' 'Select output file: Pixel Fit Values' ); fID = fopen(fullfile(dataPath2, dataFile2), 'w' ); fprintf(fID, 'Pixel Fit Values\n' ); titleVector = [ '\t' ]; fprintf(fID, 'Pixel\t' ); for index = 1:length(eyeList) fprintf(fID, [ 'Eye num2str(eyeList(index)) '\t\t\t\t' ]); titleVector = [titleVector 'Pre\tPost\tRaw Diff.\tPre Fit/Post Raw Diff.\t' ]; end fprintf(fID, '\n' ); fprintf(fID, [titleVector '\n' ]); for index = 1:1024; fprintf(fID, [num2str(index) '\t' ]); for index2 = 1:length(eyeList) fprintf(fID, [num2str(preMean(index,index2)) '\t' ... num2str(postMean(index,index2)) '\t' ... num2str(rawDiff(index,index2)) '\t' ... num2str(theDiff(index,index2)) '\t' ]); end fprintf(fID, '\n' ); end fclose( 'all' )

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152 [dataFile3, dataPath3] = ... uiputfile( '*.txt' 'Select output file: Parabolic Fit Parameters' ); fID = fopen(fullfile(dataPath3, dataFile3), 'w' ); fprintf(fID, 'Parabolic Fit Parameters\n' ); fprintf(fID, 'y = a*x^2 + b*x + c\n' ); fprintf(fID, 'Eye\tPre\n' ); fprintf(fID, '\ta\tb\tc\tRsquare\n' ); for index = 1:length(eyeList) fprintf(fID, [num2str(eyeList(index)) '\t' ... num2str(pPre(1,index)) '\t' num2str(pPre(2,index)) '\t' ... num2str(pPre(3,index)) '\t' num2str(RsquarePre(index)) '\n' ]); end fclose( 'all' )

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153 APPENDIX C EQUIPMENT LISTING This appendix includes the manufacturers and model numbers for essential components implemented in the experiments outlined in Chapters 2. Table C-1: Manufacturers and Model numbers of experimental components Component Manufacturer Model ArF Excimer Laser TuiLaser FTLasikStar ArF Excimer Laser GAM Laser, Inc. EX5 Nd:YAG Laser Big Sky Technologies, Inc. Ultra UL120111 Scan Cube Scanlab 38966 Phototube Detectors Hama matsu Photonics R1193U-02 L2 Focusing Lens Lambda Research Optics, Inc. 24459 193-nm Interference Filter Melles Griot 03FIU 101 193-nm Beam Homogenizer ME MS Optical Custom Part Delay Generators Stanford Research Systems, Inc. DG535 355-nm Edge Filter Semrock Razoredge LP01-355RU-25 Digital Oscilloscope LeCroy Waverunner LT372 iCCD Camera Andor Technology DH734-25F-03 iCCD Camera Princeton Instruments, Inc. 7397-0072 Mass Spectrometer Hiden Analytical Ltd. HPR 20 White-Light Interferometer Zygo NewView

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154 LIST OF REFERENCES 1. LASIK Surgery News. LASIK by the Numbers [updated 3 July 2008; cited 24 July 2008]. Available from http://lasiksurgerynews.com/news/eye-vision-statistics.shtml. 2. S. D. Klyce and R. W. Beuerman, Str ucture and function of the cornea, in The Cornea (Butterworth-Heinemann, Boston, 1998). 3. M. H. Niemz, Laser-Tissue Interactions (Springer, New York, 1996). 4. E. W. Kornmehl, R. K. Mal oney, and J. M. Davidorf, LASIK: A Guide to Laser Vision Correction (Addicus Books, Nebraska, 2006). 5. A. Vogel and V. Venugopalan, Mechanism s of pulsed laser ablation of biological tissues, Chem. Rev. 103, 577 (2003). 6. S. Lerman, The cornea, in Radiant Energy and the Eye (Macmillan, New York, 1980). 7. C. Boote, S. Dennis, R. H. Newton, H. Puri and K. M. Meek, Collagen fibrils appear more closely packed in the prepupillary corn ea: Optical and biomechanical implications, Invest. Ophth. Vis. Sci. 44, 2941 (2003). 8. B. T. Fisher and D. W. Hahn, Measurement of small-signal absorption cross section of collagen for 193-nm excimer laser light and the role of collagen in tissue ablation, Appl. Optics 43, 5443 (2004). 9. J. Pearce and S. Thomsen, Rate process analysis of thermal damage, in OpticalThermal Response of Laser-Irradiated Tissue (Plenum Press, New York, 1995). 10. V. Venugopalan, N. S. Nishioka, and B. B. Miki c, The effect of laser parameters on the zone of thermal injury produced by laser abla tion of biological tissu e, J. Biomech. Eng. T ASME 116, 62 (1994). 11. M. B. McDonald and D. Chitkara, Princ iples of excimer laser photoablation in The Cornea (Butterworth-Heinemann, Boston, 1998). 12. E. E. Manche, J. D. Carr, W. W. Haw, a nd P. S. Hersh, Excimer laser refractive surgery, Western J. Med. 169, 30 (1998). 13. R. A. Eiferman, K. P. ONeill, D. R. Forgey, and Y. D. Cook, Excimer laser photorefractive keratectomy for myopia: Si x-month results, Refract. Corneal Surg. 7, 344 (1991). 14. B. T. Fisher and D. W. Hahn, Developme nt and numerical solution of a mechanistic model for corneal tissue ablation with the 193 nm argon fluoride excimer laser, J. Opt. Soc. Am. A. 24, 265 (2007).

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156 28. B. T. Fisher, K. A. Masiello, M. H. Goldstei n, and D. W. Hahn, A ssessment of transient changes in corneal hydration using confocal Raman spectroscopy, Cornea 22, 363 (2003). 29. J. R. Jimnez, R. G. Anera, L. Jimnez del Barco, and E. Hita, Influence of laser polarization on ocular refract ive parameters after refract ive surgery, Opt. Lett. 29, 962 964 (2004). 30. J. R. Jimnez, R. G. Anera, L. Jimnez del Barco, E. Hita, and F Prez-Ocn, Correction factor for ablation algorithms used in corn eal refractive surgery with gaussian-profile beams, Opt. Express 13, 336 (2005). 31. J. R. Jimnez, F. Rodriquez-Marin, R. G. Anera, and L. Jimnez, Deviations of Lambert-Beer's law affect corneal refractive parameters after refractive surgery, Opt. Express 14, 5411 (2006). 32. R. G. Anera, J. R. Jimenez, L. Jimenez del Barco, and E. Hita, Changes in corneal asphericity after laser refr active surgery, including re flection losses and nonnormal incidence upon the anterior cornea, Opt. Lett. 28, 417 (2003). 33. R. G. Anera, C. Villa, J. R. Jimenez, R. Gutierrez, and L. Jimenez del Barco, Differences between real and predicted corn eal shapes after aspheri cal corneal ablation, Appl. Optics 44, 4528 (2005). 34. USA Eyes. The Odds You Will See 20/20 After Lasik, All-Laser Lasik, PRK, LASEK, EpiLasik, CK, P-IOL, RLE, etc [updated 8 April 2006; cited 24 July 2008]. Available from http://www.usaeyes.org/faq/subjects/odds.htm. 35. T. Moller-Pedersen, H. D. Cavanagh, W. M. Petroll, and J. V. Jester, Stromal wound healing explains refractive instability and haze development after photorefractive keratectomy, Ophthalmology 107, 1235 (2000). 36. J. V. Jester, T. Moller-Pedersen, J. Huang, C. M. Sax, W. T. Kays H. D. Cavanagh, W. M. Petroll, and J. Piatigorsky, The cellula r basis of corneal tran sparency: Evidence for corneal crystallins, J. Cell Sci. 112, 613 (1999). 37. K. B. Kim, L. M. Shanyfelt, and D. W. Ha hn, Analysis of dense-medium light scattering with applications to corneal tissue: Experi ments and Monte Carlo simulations, J. Opt. Soc. Am. A. 23, 9 (2006). 38. USA Eyes. Enhancement Surgery For Lasik, PRK, LASEK, Epi-Lasik, CK, etc. [updated 8 April 2006; cited 24 July 2008]. Available from http://www.usaeyes.org/faq/subjects/enhancement.htm. 39. R. Srinivasan, Kinetics of the ablative phot ocomposition of organic polymers in the far ultraviolet (193 nm), J. Vac. Sci. Technol. 1, 923 (1983).

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161 BIOGRAPHICAL SKETCH Leia Shanyfelt was born in Florida, in 1978. She was raised in Florida, graduating from Citrus High School in 1996. She graduated from the University of Florida in 2001, with her Master of Science degree in mechanical engineer ing, specializing in thermal science. Her thesis topic used experimental and theoretical models of light scattering in dens e structures to better understand haze formation in human eyes resul ting from photorefractive surgery. She then moved to South Carolina to work at the Departme nt of Energys Savannah River Site. There, she was employed by Bechtel, a private construction fi rm, as a design engineer. She returned to the University of Florida, in 2005. In 2008, she rece ived her PhD under the advisement of Dr. David Hahn. Leia is the daughter of Phillip and Nanc y Coffey and the wife of Daniel Shanyfelt.