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A Study of the Sub-Ablative Interaction between 193-Nm Arf Excimer Laser Radiation and Peptide Bonds in Selected Dipeptides

Permanent Link: http://ufdc.ufl.edu/UFE0043844/00001

Material Information

Title: A Study of the Sub-Ablative Interaction between 193-Nm Arf Excimer Laser Radiation and Peptide Bonds in Selected Dipeptides
Physical Description: 1 online resource (69 p.)
Language: english
Creator: Loper, Kristofer H
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: bonds -- excimer -- laser -- peptide -- perturbation -- sub-ablative
Mechanical and Aerospace Engineering -- Dissertations, Academic -- UF
Genre: Mechanical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Photorefractive surgery of the eye, which involves the ablation of corneal tissue using 193-nm radiation to correct optical properties (near-sighted, far-sighted, astigmatism), is increasing in popularity. Unfortunately, there is not a well-defined model that can accurately predict the ablation rates of this procedure. In other applications, 193-nm radiation may be used as an optical probe of tissues, for example to detect skin cancers. The cornea and other surface tissues are made up of water and collagen, with the latter made of amino acids bonded together by peptide bonds in long strands. It is reported that the peptide bond is the main absorber of energy in the cornea at 193 nm. Therefore, it is desirable to build a fundamental understanding of the interactions between 193-nm laser radiation and peptides, including peptide bonds. The current study focuses on the interaction of sub-ablative 193-nm ArF excimer laser radiation with peptide bonds in selected dipeptides. The dipeptides Gly-Gly, Ala-Gly, and Gly-Pro were tested due to their common occurrence in biological tissues. It was found that the perturbation of the absorption cross-sections of isolated amino acids (Gly, Ala, and Pro) by the excimer laser was negligible over the range of pulse energies studied. However, for the dipeptides, as the number of photons interacting with the dipeptides increased, the absorption coefficient of the dipeptides decreased, suggesting that the primary absorbers (i.e. peptide bonds) were broken. This reinforces the assumption that the peptide bonds are the primary chromophore, given that the peptide bond is the only difference between the isolated amino acids and the dipeptides. A relationship between the number of photons acting on the dipeptides and the number of broken peptide bonds was formulated , and it was found that Gly-Gly, Ala-Gly, and Gly-Pro required 5.8, 6.0, and 2.5 photons to break one peptide bond, respectively. The difference in these values was attributed to the difference in molecular structure between Gly-Pro (which has a cyclic structure near the peptide bond) and the other two dipeptides.
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 Kristofer H Loper.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Hahn, David W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043844:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043844/00001

Material Information

Title: A Study of the Sub-Ablative Interaction between 193-Nm Arf Excimer Laser Radiation and Peptide Bonds in Selected Dipeptides
Physical Description: 1 online resource (69 p.)
Language: english
Creator: Loper, Kristofer H
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: bonds -- excimer -- laser -- peptide -- perturbation -- sub-ablative
Mechanical and Aerospace Engineering -- Dissertations, Academic -- UF
Genre: Mechanical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Photorefractive surgery of the eye, which involves the ablation of corneal tissue using 193-nm radiation to correct optical properties (near-sighted, far-sighted, astigmatism), is increasing in popularity. Unfortunately, there is not a well-defined model that can accurately predict the ablation rates of this procedure. In other applications, 193-nm radiation may be used as an optical probe of tissues, for example to detect skin cancers. The cornea and other surface tissues are made up of water and collagen, with the latter made of amino acids bonded together by peptide bonds in long strands. It is reported that the peptide bond is the main absorber of energy in the cornea at 193 nm. Therefore, it is desirable to build a fundamental understanding of the interactions between 193-nm laser radiation and peptides, including peptide bonds. The current study focuses on the interaction of sub-ablative 193-nm ArF excimer laser radiation with peptide bonds in selected dipeptides. The dipeptides Gly-Gly, Ala-Gly, and Gly-Pro were tested due to their common occurrence in biological tissues. It was found that the perturbation of the absorption cross-sections of isolated amino acids (Gly, Ala, and Pro) by the excimer laser was negligible over the range of pulse energies studied. However, for the dipeptides, as the number of photons interacting with the dipeptides increased, the absorption coefficient of the dipeptides decreased, suggesting that the primary absorbers (i.e. peptide bonds) were broken. This reinforces the assumption that the peptide bonds are the primary chromophore, given that the peptide bond is the only difference between the isolated amino acids and the dipeptides. A relationship between the number of photons acting on the dipeptides and the number of broken peptide bonds was formulated , and it was found that Gly-Gly, Ala-Gly, and Gly-Pro required 5.8, 6.0, and 2.5 photons to break one peptide bond, respectively. The difference in these values was attributed to the difference in molecular structure between Gly-Pro (which has a cyclic structure near the peptide bond) and the other two dipeptides.
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 Kristofer H Loper.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Hahn, David W.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-06-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043844:00001


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1 A STUDY OF THE SUB ABLATIVE INTER ACTION BETWEEN 193 nm ArF EXCIMER LASER RADIATION AND PEPTIDE BONDS IN SELECTED DIPEPTIDES By KRISTOFER HOWELL LOPER A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Kristofer Howell Loper

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3 ACKNOWLEDGMENTS I thank the chair of my supervisory committee for his continued mentoring and advising throughout my research and the members of my supervisory committee for taking the time to analyze and critique my thesis. I thank my parents for their loving encouragement, which motivated me to complete my study. I tha nk m y fianc e for her patience and her support during the tougher times of my study.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 3 LIST OF TABLES ................................ ................................ ................................ ........................... 5 LIST OF FIGURES ................................ ................................ ................................ ......................... 6 ABSTRACT ................................ ................................ ................................ ................................ ..... 9 CHAPTER 1 BACKGROUND ................................ ................................ ................................ .................... 11 Excimer Laser Ablation ................................ ................................ ................................ .......... 11 UV P hotochemistry ................................ ................................ ................................ ............. 14 Differential Laser Induced Perturbation Spectroscopy (DLIPS) ................................ ........... 16 2 MATERIALS AND METHODS ................................ ................................ ........................... 18 Lase r Setup and Materials ................................ ................................ ................................ ...... 18 Sample Cell ................................ ................................ ................................ ............................. 20 Experimental Methods ................................ ................................ ................................ ............ 22 3 EXPERI MENTAL RESULTS ................................ ................................ ............................... 33 Absorption Cross Section Measurements ................................ ................................ ............... 33 Amino Acid Solutions ................................ ................................ ................................ ............ 33 Dipeptide Solutions ................................ ................................ ................................ ................ 33 4 ANALYSIS AND DISCUSSION ................................ ................................ .......................... 57 Non Perturbation Experiments ................................ ................................ ............................... 57 Perturbation Experiments ................................ ................................ ................................ ....... 58 5 CONCLUSIONS AND FUTURE WORK ................................ ................................ ............. 62 APPENDIX: ERROR ANALYSIS ................................ ................................ ................................ 64 LIST OF REFERENCES ................................ ................................ ................................ ............... 67 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ......... 69

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5 LIST OF TABLES Table page 2 1 Pulse energies and fluences of the perturbation beam. ................................ ...................... 28 2 2 Characteristics of the amino acids and the dipeptides. ................................ ...................... 28 3 1 The Absorption Cross sections of the individual amino acids, their dipeptides, and the peptide bond in the dipeptides are recorded. ................................ ................................ 38 3 2 The slopes of the Absorption Coefficient vs. Number of Photons plots for Glycine Glycine. ................................ ................................ ................................ .............................. 55 3 3 The slopes of the Absorption Coefficient vs. Number of Photons plots for Glycine Pro line ................................ ................................ ................................ ................................ 55 3 4 The slopes of the Absorption Coefficient vs. Number of Photons plots for Alanine Glycine. ................................ ................................ ................................ .............................. 55 3 5 The number of photons it takes to break one peptide bond for each of the dipeptides. .... 56 A 1 Beam energy fluctuation data for the 193 nm excimer laser. ................................ ............ 66

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6 LIST OF FIGURES Figure page 1 1 Example amino acid sequence derived from collagen ................................ ....................... 17 1 2 Fluorescence spectra recorded from C450/BBQ thin films before (Pre) and after (Post) exposure to 250 pulses from the 193 nm perturbation laser.. ................................ 17 2 1 Schematic of the laser setup. ................................ ................................ .............................. 29 2 2 Path of the laser beam through the attenuating system ................................ ...................... 29 2 3 Schematic of the sample cell ................................ ................................ .............................. 30 2 4 Schematics of the molecular structures of Glycine, Alanine, and Proline amino acids.. ................................ ................................ ................................ ................................ 31 2 5 Schematics of the molecular structures of Glycine Glycine, Glycin e Proline, and Alanine Glycine d ipeptides. ................................ ................................ .............................. 31 2 6 Example of how the amino acids bond together. ................................ ............................... 32 2 7 Example of the transmitted and incident signal that appeared on the oscilloscope ........... 32 3 1 Plot o f Absorption Coefficient vs. Number Density for Glycine ................................ ...... 35 3 2 Plot of Absorption Coefficient vs. Number Density for Alanine. ................................ ..... 35 3 3 Plot of Absorption Coefficient vs. Number Density for Proline. ................................ ...... 36 3 4 Plot of Absorption Coefficient vs. Number Density for Glycine Glycine. ....................... 36 3 5 Plot of Absorption Coefficient vs. Number Density for Alanine Glycine.. ...................... 37 3 6 Plot of Absorpti on Coefficient vs. Number Density for Glycine Proline.. ....................... 37 3 7 Absorption data plotted as a function of the number of photons int eracting with the Glycine solution at a beam energy of 1.00 mJ/pulse ................................ ......................... 38 3 8 Absorption data plotted as a function of the number of photons interacting with the Glycine solution at a beam energy of 1.50 mJ/pulse. ................................ ........................ 39 3 9 Absorption data plotted as a function of the number of photons interacting with the Glycine solution at a beam energy of 2.00 mJ/pulse ................................ ......................... 39 3 10 Absorption data plotted as a function of the number of photons interacting with the Glycine solution at all beam energies. ................................ ................................ ............... 40

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7 3 11 Absorption data plotted a s a function of the number of photons interacting with the Alanine solution at a beam energy of 1.00 mJ/pulse. ................................ ........................ 40 3 12 Absorption data plotted as a function of the number of photons interacting with the Alanine solution at a beam energy of 1.50 mJ/pulse. ................................ ........................ 41 3 13 Absorption data plotted as a function of the number of photons interacting with the Alanine solution at a beam energy of 2.00 mJ/pulse. ................................ ........................ 41 3 14 Absorption data plotted as a function of the number of photons interacting with the Alanine solution at all beam energies. ................................ ................................ ............... 42 3 15 Absorption data plotted as a function of the number of photons interacting with the Proline solution at a beam energy of 1.00 mJ/pulse ................................ .......................... 42 3 16 Absorption data plotted as a function of the number of photons interacting with the Proline solution at a beam energy of 1.50 mJ/pulse ................................ .......................... 43 3 17 Absorption data plotted as a function of the number of photons interacting with the Proline solution at a beam energy of 2.00 mJ/pulse ................................ .......................... 43 3 18 Absorption data plotted as a function of the number of photons interacting with the Proline solution at all beam energies ................................ ................................ ................. 44 3 19 Absorption data plotted as a function of the number of photons interacting with the Glycine Glycine solution at a beam energy of 0.99 mJ/pulse. ................................ .......... 45 3 20 Absorption data plotted as a function of the number of photons interacting with the Glycine Glycine solution at a beam energy of 1. 19 mJ/pulse. ................................ .......... 45 3 21 Absorption data plotted as a function of the number of photons interacting with the Glycine Glycine solution at a beam energy of 1.39 mJ/pulse. ................................ .......... 46 3 22 Absorption data plotted as a function of the number of photons interacting with the Gly cine Glycine solution at a beam energy of 1.60 mJ/pulse ................................ ........... 4 6 3 23 Absorption data plotted as a function of the number of photons in teracting with the Glycine Glycine solution at a beam energy of 1.87 mJ/pulse. ................................ .......... 47 3 24 Absorption data plotted as a function of the number of photons interacting with the Glycine Glycine solution at all beam energies ................................ ................................ .. 47 3 25 Absorption data plotted as a func tion of the number of photons interacting with the Alanine Glycine solution at a beam energy of 1.00 mJ/pulse. ................................ .......... 48 3 26 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at a beam energy of 1.20 mJ/pulse ................................ ........... 48

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8 3 27 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at a beam energy of 1.40 mJ/pulse. ................................ .......... 49 3 28 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at a beam energy of 1.60 mJ/pulse ................................ ........... 49 3 29 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at a beam energy of 1.80 mJ/pulse. ................................ .......... 50 3 30 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at a beam energy of 2.00 mJ/pulse. ................................ .......... 50 3 31 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at all beam energiest. ................................ ................................ 51 3 32 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at a beam energy of 1.00 mJ/pulse ................................ ............ 51 3 33 Absorption data plotted as a function of the number of photons interacting with the Glycine Proli ne solution at a beam energy of 1.20 mJ/pulse ................................ ............ 52 3 34 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at a beam energy of 1.40 mJ/pulse. ................................ ........... 52 3 35 Absorption data plotted as a function of the number o f photons interacting with the Glycine Proline solution at a beam energy of 1.60 mJ/pulse. ................................ ........... 53 3 36 Absorption data plotted as a fu nction of the number of photons interacting with the Glycine Proline solution at a beam energy of 1.78 mJ/pulse ................................ ............ 53 3 37 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at a beam energy of 2.04 mJ/pulse. ................................ ........... 54 3 38 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at all beam energies ................................ ................................ ... 54 3 39 Representative plot of Absorption Co efficient vs. Number of Photons ............................ 56

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science A STUDY OF THE SUB ABLATIVE INTERACTION BETWEEN 193 nm ArF EXCIMER LASER RADI ATION AND PEPTIDE BONDS IN SELECTED DIPEPTIDES By Kristofer Howell Loper December 2011 Chair: David W. Hahn M ajor: Mechanical Engineering Photorefractive surgery of the eye, which involves the ablation of corneal tissue using 193 nm radiation to correc t optical properties (near sighted, far sighted, astigmatism), is increasing in popularity. Unfortunately, there is not a well defined model that can accurately predict the ablation rates of this pr ocedure. In other applications, 193 nm radiation may be used as an optical probe of tissues, for example to detect skin cancers. The cornea and other surface tissues are made up of water and collagen, with the latter made of amino acids bonded together by peptide b onds in long strands. It is reported that the peptide bond is the main absorber of energy in th e cornea at 193 nm. Therefore, it is desirable to build a fundament al understanding of the interactions between 193 nm laser radiation and peptide s, including pe ptide bonds. The current study focuses on the interaction of sub ablative 193 nm ArF excimer laser radiation with peptide bonds in selected dipeptides. The dipeptides Gly Gly, Ala Gly, and Gly Pro were tested due to their common occurrence in biological ti ssue s It was found that the perturbation of the absorption cross section s of isolated amino acids (Gly, Ala, and Pro) by the excimer laser was negligible over the range of pulse energies studied. However, for the dipeptides, as the number of photons inter acting with the dipeptides increased, the absorption coefficient of the dipeptides decreased, suggesting that the primary absorbers ( i.e. peptide bonds) were broken. This

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10 reinforces the assumption that the peptide bond s are the primary chromophore, given t hat the peptide bond is the only difference between the isolated amino acids and the dipeptides. A relationship between the number of photons acting on the dipeptides and the number of broken peptide bonds was formulated and it was found that Gly Gly, Al a Gly, and Gly Pro required 5.8, 6.0, and 2.5 photons to break one peptide bond, respectively. The difference in these values was attributed to the difference in molecular structure between Gly Pro (which has a cyclic structure near the peptide bond) and t he other two dipeptides.

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11 CHAPTER 1 BACKGROUND 193 nm ArF excimer laser based ablation has become the most widely used method for correcting refractive errors of the cornea in refractive surgery (Razhev 2009). These systems are continually evolving to improve precision and accuracy and to provide more desirable refractive results. To optimize these procedures a complete understanding of the ablation process is required. Unfortunately, the fundamentals of corneal ablation are not yet completely understood. Toward such an understanding, t he focus of this thesis will be on the fundamental relatio n between the ArF excimer laser (193 nm) and amino acids found in corneal collagen, more specifically, the peptide bonds that connect these amino acids. An additional objective of this thesis is to expand upon a relatively new diagnostic scheme know as Dif ferential Laser Induced Perturbation Spectroscopy, or D LIPS, that utilizes the interaction of the 193 nm laser with biological matrices. Excimer Laser Ablation There has been a considerable amount of literature addressing the physics and mechanisms of exci mer laser tissue ablation (Kitai 1991, Paltauf 2003, Vogel 2003, Razhez 2009). This literature focuses on tissue ablated by UV radiation (190 250 nm). Razhez et al. discuss the thermal relationship between UV radiation, at 193 nm and 223 nm, and the corn eal surface of the eye. It was discovered that the temperature of the corneal surface increased up to 11 C during the ablation process and that this increase in temperature changes the optical properties of the cornea and possibly causes post operative complications like hazing (2009). Kitai et al. discuss the physics of UV laser cornea ablation. They state that UV laser ablation include s photochemical, thermal, and mechanical processes. When the UV laser is absorbed by the cornea the energy is transformed into heat. This heat serves two purposes. First, it causes the

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12 macromolecules in the cornea to vibrate which can result in their ultim ate breakage. Second, it causes water to boil. The expansion caused by the vaporization of water puts added mechanical stresses on the macromolecules which can also lead to their breakage (1991). On the other hand, the UV laser photons may directly fractur e bonds. All of these breakages result in fragments of the original macromolecule, and these fragments require a significantly larger volume to occupy for equilibrium. Due to this, the fragments get expelled (ablated) from the tissue. Mechanical stresses a re also apparent when the tissue attempts to reconfigure itself to an equilibrium state after the ablated material is expelled (Vogel 2003). The cornea is a collageni c tissue which is composed of 78% water It is reported that water displays significant a bsorption at 170 nm; however, water displays negligible absorption at 193 repeating pattern of amino acids. Glycine (Gly), Proline (Pro), and H ydroxyproline (Hyp) are the three most common a mino acids (Glycine is found in roughly every third amino acid but Proline and H ydroxyproline are sometimes replaced with other amino acids) which repeat themselves in this pattern, and this sequence can be represented as Gly P ro Hyp, as depicted in F igur e 1 1 These amino acids are bonded together by a peptide bond (C N). The peptide bonds connecting the adjoining amino acids are responsible for 96% of the absorption by collagen of the excimer laser beam at a wavelength of 193 nm (Fisher 2004). For this r eason, a fundamental relationship between 193 nm ArF excimer laser radiation and peptide bonds must be explored. To date, most resources describe the interaction between corneal tissue and excimer laser radiation to behave according to the Beer Lambert la w, which is described in the following equation: I(x) = I O exp( ) ( 1 1)

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13 In this equation I(x) is the intensity of the radiation after penetrating to a depth x (usually measured in cm) in the tissue, I O is the intensity of the laser radiation on the surf ace of the tissue ( x = 0), and (cm 1 ) is the absorption coefficient which for 193 nm radiation has been reported as 16000 cm 1 Unfortunately, studies have shown that the Beer Lambert law does not hold for a wide range of tissue incident intensities which suggest that a dynamic model is necessary to predict laser penetration and ultimately laser ablation (Jimenez 2006, Fisher 2007). Other analytical models describing the relationship between collagen and excimer laser ablation have been developed. J imenez et al. suggest that ablation rate data (provided by Kruger et al. 1985) is fit much better by a quadratic relat ion rather than the usual relation generated by the Beer Lambert law. This conclusion suggests that there are models that can be produced to better predict tissue ablation rates and improve corrective refractive surgery (2006). Sutcliffe and Srinivasan introduced a dynamic model which states that photofragmentation is negligible below an absorbed photon flux threshold as well as a critical density of broken bonds threshold. In other words, for ablation to occur the irradiated sample must absorb photons per unit time above a defined threshold and the sample must reach a critical density of broken bonds above another defined threshold. Overall this defines an ablation threshold for which ablation does not occur below this threshold and ablation does occur above this threshold (1986). Srinivasan et al. from the electronically excited state of the bonds rather than from the ground electronic state of the bonds, and mechanical force then cleaves the tissue. This method also takes into account a dly below this threshold fluence (1986). Tokarev et al. suggest that an analytical thermal model, neglecting photodecomposition concepts, can be used to predict UV laser ablation. The model takes into account the concepts of

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14 laser beam shielding by the abl ated particle plume, the variation of optical properties due to temperature change, and the thermal conductivity effect on ablation kinetics (1995). Vogel and Venugopalan compare Blow off and Steady State models and they describe steady state vaporization models and thermomechanical models that could be applied to the ablation process et al. describe a model that involves a four level excitation relaxation excitation process, for which each level has a different absorption cross section (1996). Fisher and Hahn developed a dynamic ablation model that m akes use of the differential form of the Beer Lambert Law along with dynamic absorption coefficients. They concentrate their interest on four species: (1) collagen, (2) water, (3) a transien t absorber formed by the interaction of the laser photons with the collagen amino acids, and (4) a stable non absorber formed by the interaction of the transient absorber with surrounding water. Each of these species has a specific absorption cross section and number density and these are used to describe the general corneal tissue absorption coefficient (2007). As demonstrated from the above resources, there are a lot of models that have been proposed that attempt to predict tissue ablation rates. Some mo dels focus on the thermal and mechanical relationship between excimer laser radiation and corneal tissue and other models focus on the photochemistry between the laser radiation and the tissue. Due to this variation, a blation is removed from the picture i n this work, and the question is asked : What happens before the ablation threshold is reached? UV Photochemistry Sub ablative ArF excimer laser radiation is not extensively researched because to date edure can be useful because it does not involve the complex ablation process. As mentioned above, the ablation process includes thermal, mechanical, and photochemical processes. Vogel and Venugopalan believed that the energy absorbed from sub ablative radi ation by the corneal tissue only results in a photothermal

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15 reaction (2003). However, it was recently discovered that perturbations of collagen caused by sub ablative radiation were produced solely by a photochemical reaction and that thermal reactions wer e negligible. This same study also discovered that the peptide bond is the primary chromophore in the case of sub ablative perturbation by 193 nm and 355 nm radiation in the energy range of 0.55 to 1.2 mJ/pulse (Shanyfelt 2008). Based on the above informat ion, this sub ablative procedure allows one to study the photochemical reaction occurring between 193 nm laser radiation and peptide bonds without worrying about thermal or mechanical processes (they are assumed to be negligible). Gorner and Nikogosyan hav e produced many papers (1994, 1995, 1998, 1999) tha t focus on the photoche mistry of some amino acids and dipeptides at various UV radiation s (193 266 nm) They report the quantum yields (the ratio of an event happening to the number of photons absorbed) of photoionization, photodecomposition, and peptide bond scission for these amino acids and dipeptides. Photoionization occurs when an amino acid or dipeptide absorbs the energy from the photons and separates into an amino acid cation, AA (+) and an anion, e aq ( ) This resulting cation can then deprotonate or react with water (refer to Scheme 1 in Nikogosyan and Gorner, 1995). The resulting anion reacts with laser induced protons and it was assumed that they do not contribute to photodecomposition. They also repor ted that the sequence of amino acids in a dipeptide can cause different quantum yields of photodecomposition and peptide bond scission. For example, Gly Pro has a greater quantum yield of peptide scission than does Pro Gly. Looking at F igure 2 5 this may structure to the peptide bond. The overall result derived from these papers was that 60% of all the illuminated peptide bonds of corneal collagen were subject to splitting by one laser pulse.

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16 This was an extrapolated result, as hinted at by the title (1999), and actual data to support this conclusion has yet to be discovered. Differential Laser Induced Perturbation Spectroscopy (DLIPS) Differential Laser Induced Perturbation Spectroscopy (DLIPS) is a relatively new diagnostic technique. In a 2010 study, Smith et al. used this diagnostic tool to determine changes in laser dyes, Coumarin 450 (C13H16NO2) and BBQ (C48H66O2), after they were perturbed with an ArF excimer laser (193 nm). The laser deliver ed 250 pulses with energy of 100 J/pulse to the dyes. This energy is well below the ablation threshold, and therefore, only the molecular structure of the dyes was disrupted via direct bond breakage. To obtain a pre and post perturbation spectrum a Nd:YA G laser (355 nm), coaxially aligned with the excimer laser, was used to excite the dyes and cause them to fluoresce. These fluorescent spectra (both pre and post perturbation) were recorded and the difference between these two spectra was calculated. A s am ple of this method is shown in F igure 1 2. From this difference spectrum the authors discovered that the optical properties, in this case fluorescence emission, were altered due to the perturbation shots delivered by the excimer laser (Smith 2011). For t he purposes of this study, the same general technique will be used, except the primary optical property we will be focusing on is the transmissivity of the sample.

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17 Figure 1 1 Example amino acid sequence derived from collagen. Glycine accounts for every third amino acid. Proline and Hydroxyproline are the next two most common amino acids present but they are sometimes replaced by other amino acids. Figure 1 2 Fluorescence spectra recorded from C450/BBQ thin films before (Pre) and after (Post) exposure to 250 pulses from the 193 nm perturbation laser. Both spectra have the same scale, and the lower spectrum corresponds to the difference between the post and pre perturbation spectra. Borrowed with permission, from reference: Smith 2010.

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18 CHAPTER 2 MATERIALS AND METHOD S Laser Setup and Materials In order to study the relation between peptide bonds connecting the amino acids and sub ablative laser radiation, transmission experiments were implemented. The same laser, the ArF 193 nm excimer l aser, is used both as the analytical probe laser and as the perturbation laser. The schematic o f the setup is shown in F igure 2 1 The beam produced by the excimer laser passes through an aperture and then interacts with a glass quartz window aligned at a 45 O angle; about 95% of the beam energy transmits through the glass quartz and about 5% of the beam energy is reflected 90 O by the glas s quartz. The larger energy beam, ~95% energy, is considered the perturbation beam and the smaller energy beam is conside red the probe beam. Both of these beams then pass through a shield, which is a slab of low reflective black cardboard with holes cut into it. The shield was necessary because the upper detector, shown in the schematic, was detecting stray bea m energy from the laser and surrounding reflective surfaces. The perturbation beam then passes through a shutter (Laser Safety Shutter CX 2450B Interlock Controller) which allows manual on/off control of the perturbation beam; this functionality will be discussed later on in this chapter. Next, the perturbation beam travels through a system that controls the energy of the perturbation beam experienced by the sample 193 nm dichro ic lens es, which at a 45 O angle to the beam reflects ~98% of the beam energy and ~2% of the energy is transmitted through it while at a 0 O angle to the beam this mirror transfers ~98% of the beam energy and reflects ~2% of the energy; these are the properties for all o bj ects labeled as F igure 2 1 ) that are aligned with supplementary angles to maintain the path direction of the beam, as shown i n F igure 2 2 The amount of beam energy that is transmitted

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19 1 seen in the figure. This system was covered by a black reflective beam energy from this system. After this, the perturbation beam passes through another aperture to reduce i ts area and achieve a smaller beam diameter. Finally, this beam is reflected by a mirror and interacts with the sample cell. The perturbation beam diamete r is 5.0 mm and the pulse energies and fluences are recorded in T able 2 1 as well as the number of p hotons per pulse and the photon flux. The beam profile is approximately a top hat profile (i.e. uniform fluence). Now, attention is switched to the probe beam. After passing through the shield this beam passes through another ape rture to reduce its diamet er to 4 mm, a smaller value than that of the perturbation beam 5 mm Next, the probe beam interacts with a second mirror. The energy that passes through this mirror is collected by the upper detector (Hamamatsu R1193U Phototube), s een in F igure 2 1 which transmits this signal, which was denote d as added in front of this detector to maintain signal linearity. The reflected part of the beam tr avels through the sample cell, through another mirror, and is then collected by the second detector, shown in F igure 2 1 Both detectors had 193 nm narrow line filters in front of them. The pro be beam is aligned coaxially with the perturbation beam as seen in F igure 2 3 so that the probe beam interacts with the perturbed part of the sample cell solution. The incident and transmitted signals usually vary from shot to shot (ex. the incident sig nal for the first shot may be different than the second shot under the same conditions). This variation is caused by the excimer laser itself. In order to account for this variation, the ratio of the

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20 transmitted and incident signals was calculated and thi s ratio was relatively constant from shot to shot because the differences seen by the transmitted and incident signals from shot to shot are the same. Overall, the only difference between the incident signal and the transmitted signal is that the transmitt ed signal passes through the sample solution. Due to these two facts, one is able to determine the absorption properties of the sample cell using the ratio of the two signals. This will be discussed in more detail later in this chapter. Sample Cell There w ere seven different solutions that were used during this experiment, all of which included de ionized (DI) water: Glycine (Gly), Proline (Pro), Alanine (Ala), Glycine Glycine (Gly Gly), Glycine Proline (Gly Pro), Alanine Glycine (Ala Gly), and the seventh was just DI water. These amino acids and their dipeptides were chosen because they commonly appear in the collagen structure of the cornea (with the exception of Gly Gly) and they were readily available. Schematics of all molec ules listed above are shown i n F igures 2 4 and 2 5 and T able 2 2 includes some of their characte ristics. Table 2 2 also make up that dipeptide (Ex. Difference = M Gly Pro M Gly M Pro ). For each one the difference was 18.02 g/mol, the mola r mass of a water molecule. During the bonding of two amino acids, an OH species from one amino acid and an H species from the other amino acid (boxed species in F igure 2 4 ) bond together to form a water molecule, which is expelled from t he dipeptide, and this is why one see s this differen ce in molecular masses. F igure 2 6 shows an example of th is bonding process between two G lycine amino acids. Concentrations of Glycine, Proline, and Alanine solutions were created using the same procedure. First, 48 mg of amino acid (AA) was dissolved in 96 mL of DI water. This solution was then divided into four equal solutions: 12 mg AA dissolved in 24 mL water (1:2). 24 mL of

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21 water was added to one of the resultant solutions, 48 mL of water was added to another, 72 mL of water was added to another, and nothing was added to the last solution. This resulted in four concentrations: 12 mg AA: 24 mL H 2 O (1:2), 12 mg AA: 48 mL H 2 O (1:4), 12 mg AA: 72 mL H 2 O (1:6), 12 mg AA: 96 mL H 2 O (1:8). Concentrations of Glycine Glycine, Glycine Proline, and Alanine Glycine (dipeptides or DP) solutions were created the same way as the above solutions, but with different concentrations. The process was started with 8 mg of DP dissolved in 200 mL of water a nd divided this into four equal solutions: 2 mg DP dissolved in 50 mL water (1:25). Then, 10 mL water was added to one solution, 20 mL water to another, 30 mL water to another, and nothing was added to the last solution. This resulted in four concentration s for the dipeptides: 2 mg DP: 50 mL H 2 O (1:25), 2 mg DP: 60 mL H 2 O (1:30), 2 mg DP: 70 mL H 2 O (1:35), 2 mg DP: 80 mL H 2 O (1:40). For perturbation experiments a 1:5 (30 mg AA: 150 mL H 2 O) concentration was used for the amino acid solutions and a 1:35 (7 mg DP: 245 mL H 2 O) concentration was used for the dipeptide solutions. The amino acid solutions are roughly seven times more concentrated than the dipeptide solutions in order to decrease the transmission signals because they lack the primary chrom ophore, namely, the peptides bond. The sample cell was composed of two quartz flats, an o ring, and the specific solution selected for analysis (Figure 2 3 ). To assemble the sample cell, a quartz flat and an o ring were laid down on a flat surface (the o r ing on top of the quartz flat). Then solution was poured into this cavity until its surface level was roughly even with the height of the o ring. Finally, the second quartz flat was pushed onto this assembly, sealing the solution in between the o ring and the two quartz flats and effectively holding the sample cell together by suction. The path length

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22 of the solution was calculated by subtracting the width of the two quartz flats (width of one quartz flat equaled 0.311 cm) by the total width of the assemble d sample cell, which was measured throughout the experiment with a micrometer (accuracy 0.0013 cm) to render an average value of 0.790 cm (this valued ranged from 0.795 cm to 0.785 cm). Therefore, an average path length of 0.168 cm was used for all of our Experimental Methods First, experiments were run to find the absorption cross sections of the ami no acids and the peptide bonds. S ample cells were made out of water and the amino acids at 1:2, 1:4, 1:6, and 1:8 con centrations as discussed above. A spot on the sample cell was sampled then the cell was then rotated 120 (1/3 360 ) and another spot was sampled and finally the cell was rotated another 120 and a third and final spot was sampled The average transmis sion value from these spots was then calculated For each spot, 20 laser pulses were averaged to obtain an overall incident signal and an overall transmitted signal. Examples o f these signals are plotted in F igure 2 7 These signals were then related to o ne another using the Beer Lambert law: ( 2 1) 0 is the intensity of the incident laser energy, N is the number density of absorbers (cm 3 section of the absorbers (cm 2 as 0.168 cm). The absorption cross sect ions for all of the current work is defined at a wavelength of 193 nm, with a spectral width corresponding to the excimer laser, a value estimated to be <0.01 nm. The transmission calculated here takes into account the transmission through the amino acids, as well as the transmission through water and the quartz flats. In order to decouple

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23 the transmission through the amino acid from the transmission through the water and quartz flats a sample cell of just DI water was put through this experiment and the f ollowing equation was utilized: (2 2) total is the transmission through the sample cell that includes quartz flats, water, and amino water is the transmission through the sample amino acid is the transmission through just the amino acids. With this transmission calculated it is possible the amino acids using Equation 2 2 and the known path length. In order to determine the absorption cross sections of the amino acids the number density of the amino acids needed to be determined. The following equation was used to determine these number densities: (2 3) In this equation, N is the number density of the absorbers (cm 3 ), N A (6.022e23 mol 1 ), M is the molar m density/concentration of the amino acid in the sample solution (mg/mL). At this point, t he are known at four different concentrations. T he absorption coefficient s are then plotted versus the number density to produce a scatter plot. A best fit linear line was fitted onto this data and the slope of this line was taken as the absorption cross section of the amino acid being analyzed. The r esults of this Analysis and Discussion What follows is a step by step overview of the procedures for this part of the experiment: A DI water sample cell was created and three spots on this cell were lased with each spot

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24 receiving 20 laser pulses, and the transmission through this cell was calculated T he above step was repeated with amino acid solutions at concentrations of 1:2, 1:4, 1:6, and 1:8. Next, the absorption coefficient and the nu mber density of the amino acid for each concentration was calculated using E quations 2 2 and 2 3, respectively. Finally, these two results were plotted against one another and the absorption cross section of the amino acid was determined based on the sl ope of this plot. This procedure was then repeated for the dipeptides to obtain their absorption cross sections. Since absorption cross sections ar e additive (as demonstrated by E quation s 2 4 and 2 5) the absorption cross sections of the peptide bonds wer e calculated for each dipeptide that was studied using simple algebra: where (2 4 ) (2 5) ( 2 6) (2 7) (2 8) This process neglects the changes in peptide cross section due to the hydration process during formation. For the sec ond part of this experiment, the goal was to determine the fundamental relation between peptides bonds and the 193 nm excimer laser. There was o nly one amino acid concentration (1:5) and one dipeptide concentration (1:35) used for this part of the experiment. Therefore, for t he dipeptide concentrations, one could figure out the number of peptide bonds that were in the solution by using E quation 2 3 2 O.

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25 First, the focus here is on the procedures for the amino acid solutions. As mentioned before, a concentration of 1:5 AA:H 2 O (30 mg AA: 150 mL H 2 O) was used for these solutions. These solutions were tested at three different perturbation beam energies, and six samples of the solutions were tested for each beam energy; therefore, a total of eighteen sample cells were usin g the manual on/off shutter control that was mentioned above. The solution was put into the experienced 10 perturbation shots. Since the laser was set to a rate of 2 Hz, the number of perturbation shots that were to be applied was manually counted, and then the perturbation beam was immediately blocked using the shutter control. Then another 10 s hots of the probe beam were averaged to 72 (1/5 of 360 n the solution experienced 20 perturbation shots, followed by 10 probe 72 and the process was repeated with 30 perturbation shots, then 40 perturbation shots, and finally with 50 perturbation shots. Overall, this one sample cell was perturbed in five different spots with these spots experiencing 10 perturbation shots, 20 shots, 30, 40 and then 50 perturbation shots. After this, another sample cell was construct ed and it underwent the same procedures as the first sample cell. A DI water sample was tested before and af ter these sample cells so that E quation 2 2 could be utilized. This process was repeated until six different sample cells were tested this way. Then the beam energy was changed and the whole process repeated itself. For the amino acids there were three beam energies tested: ~1.00 mJ/pulse, ~1.50

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26 mJ/pulse, and ~2.00 mJ/pulse (all beam energies have errors of about 0.05 mJ/pulse). The goal of this e xperiment was to relate the change of the absorption coefficient to the number of photons that impinged on the solution. To determine the number of photons that are reacting with the solution the following equations were used: (2 9) (2 10) In this equation, n is the number of photons experienced by the solution, E t is the perturbation beam energy per pulse, N p is the number of pulses, and E ph is th e photon energy (as defined by E quation 2 10 for the 193 nm excimer laser; h c is the speed of light, and is the wavelength of the laser). The absorption coefficient w as calculated as before, u sing E quations 2 1 and 2 2. Plotting these two values against one another (absorption coefficient vs. number of photons) resulted in a scatter plot, to which a linear best fit line was set to. The slope of this best fit line represents the change in th e ab sorption coefficient per incident photon. The above procedure was then repeated for the dipeptide solutions, except with a few differences. The Gly Gly solution was tested at five differ ent perturbation energies (0.99 mJ/pulse, 1.19 mJ/pulse, 1.39 mJ/puls e, 1.60 mJ/pulse, and 1.87 mJ/pulse) requiring thirty samples cells. The Gly Pro solution was tested at six different perturbation energies (1.00 mJ/pulse, 1.20 mJ/pulse, 1.40 mJ/pulse, 1.60 mJ/pulse, 1.78 mJ/pulse, and 2.04 mJ/pulse) requiring thirty six sample cells. The Ala Gly solution was tested at six different perturbation energies (1.00 mJ/pulse, 1.20 mJ/pulse, 1.40 mJ/pulse, 1.60 mJ/pulse, 1.80 mJ/pulse, and 2.00

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27 mJ/pulse) requiring thirty six sample cells. The results of this experiment are discus sed in greater detail Discussion sub ablative experiments. To prove this assumption is valid, the change in temperature of the sample sol equation, Q is the maximum amount of energy absorbed by the sample solution for 50 perturbation shots at a beam energy of 2.00 mJ/pulse ~10 0 mJ; m is the mass of water in the sample cell, ~2.50 g; c is the specific heat of water, ~4.18 J/gK. This results in a change of temperature of <0.01 O C, which is small enough to assume thermal changes are negligible.

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28 Table 2 1. Pulse energies and fluences are reported in this table. The number of photons per pulse and the photon fluxes are also reported. The fluences are well below the ablation threshold for cornea which has been reported as ~50 mJ/cm 2 (Fisher 2007). Pulse Energy (mJ) Fluence (mJ/cm 2 ) Number of Photons per Pul se Photon Fluence (photons/cm 2 ) 1.0 5.2 7.75E+14 4.02E+15 1.2 6.2 9.30E+14 4.83E+15 1.4 7.3 1.09E+15 5.63E+15 1.6 8.3 1.24E+15 6.44E+15 1.8 9.3 1.40E+15 7.24E+15 2.0 10.4 1.55E+15 8.05E+15 Table 2 2. The characteristics of the amino acids and the dipeptides are recorded. The difference column is the molar mass of the dipeptide subtracted by its respective amino acids (i.e. Difference = M Gly Pro M Gly M Pro ). Molecule Molar Mass (g/mol) Difference Chemical Formula Brand Name Glycine 75.07 N/A C 2 H 5 NO 2 SIGMA G7403 Proline 115.13 N/A C 5 H 9 NO 2 Fluka 81709 Alanine 89.09 N/A C 3 H 7 NO 2 Fluka 05129 Glycine Glycine 132.12 18.02 C 4 H 8 N 2 O 3 SIGMA 50199 Glycine Proline 172.18 18.02 C 7 H 12 N 2 O 3 SIGMA G3002 Alanine Glycine 146.14 18.02 C 5 H 10 N 2 O 3 SIGMA A0878

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29 Figure 2 1 nm diachronic lenses. At 45 O these lenses reflect ~98% of the incident beam energy and only reflect ~2% of the incident beam energy when perpendicular to the incident beam. Figure 2 2 Path of the laser beam through the attenuating system. The transmission of energy through these dichroic lenses depends on the angle of the lenses to the laser beam; 1

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30 Figure 2 3 The probe beam is smaller than and aligned coaxially with the perturbation beam to ensure that the probe beam interacts with the perturbed part of the solution. The second picture shows the vertical alignment of the laser beams. They are offset from the center of the sample solution so that the sample cell can simply be rotated to get to a new spot to be tested.

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31 Figure 2 4 Schematics of the Glycine, Alanine, and Proline amino acids. The boxed H and OH species react to form a water molecule when these amino acids bind to one another. Figure 2 5 Schematics of the Glycine Glycine, Glycine Proline, and Alanine Glycine Dipeptides. The bond that connects two amino acids to one another is called the peptide bond.

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32 Figure 2 6 Example of how the amino acids bond together. This example is of Glycine bonding to another Glycine amino acid. Figure 2 7 Example of the transmitted and incident signal that appeared on the oscilloscope. The se signals were compared using E quation 2 1. This transmission was then compared to the transm ission of a water sample using E quation 2 2 to determine the transmission of the amino acid or dipeptide that was being analyzed. -1.00E-03 0.00E+00 1.00E-03 2.00E-03 3.00E-03 4.00E-03 5.00E-03 6.00E-03 7.00E-03 8.00E-03 9.00E-03 0 50 100 150 200 Intensity (a.u.) Pixel Number Transmitted Signal of Gly-Gly (1:25 concentration) Incident Signal of Gly-Gly (1:25 concentration)

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33 CHAPTER 3 EXPERIMENTAL RESULTS Absorption Cross Section Measurements Figures 3 1 3 6 are plots of the absorption coefficient vs. number density for the non perturbation experiments. The slopes of these plots aided in determining the absorption cross sections of the amino acids, the dipeptides, and the peptide bonds in these dipeptides. Ta ble 3 1 tabulates the results from F igures 3 1 3 6 The values in the plots are rounded to the nearest whole number; therefore, the values in the table are more accurate. The absorption cross sections of the peptide bonds are also reported. For these cal culations, it was assumed the absorption cross sections were additive. Amino Acid Solutions Figures 3 7 3 18 are derived from the perturbation experiments. Each one plots the absorption coefficient versus the number of photons for the amino acid being an alyzed. The beam energy that was used is also displayed in the plot. These figures show that the perturbation of the cross sections of isolated amino acids by the excimer laser has negligible effect on the absorption coefficient of these amino acids. Dipe ptide Solutions Figures 3 19 3 38 are derived from the perturbation experiments. Each one plots the absorption coefficient versus the number of photons for the dipeptide being analyzed. The beam energy that was used is also displayed in the plot. The dat a for the individual beam energy series were assembled to create one sin gle plot containing all the data for a given dipeptide species. In compiling all of the data, each set was normalized to a common initial absorbance, which accounts for slight differen ces (i.e. vertical offset) in the single energy experiments due to

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34 sample to sample variations in absolute baseline absorbance. It is noted, however, that the individual single energy experiments were all characterizes by similar slope. The slopes of the s ingle energy experim ents are tabulated in T able s 3 2 3 4 along with energ 24, 3 31, and 3 38 ) are also recorded, and they relate well with the Figure 3 39 is a representative plot of F igures 3 24, 3 31, and 3 38 (absorption coefficient vs. number of photons for the dipeptide being analyzed). This figure was used to aide in the formulation of the relationship between the number of photons and the number of broken peptide bonds. Table 3 5 summarizes the final desired result: the number of photons it takes to break one peptide bond in the specified dipeptides. One will notice t he similarity between Glycine Glycine and Alanine Glycine; whereas Glycine Proline requires less photons to break one of its peptide bonds. This table also records the number of broken bonds per incident photon and the error in the number of photons it tak es to break one peptide bond.

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35 Figure 3 1 Plot of Absorption Coeffici ent vs. Number Density for Glycine The error bars account for all errors in the variables needed to derive this plot. Figure 3 2 Plot of Absorption C oefficient vs. Number Density for Alanine. The error bars account for all errors in the variables needed to derive this plot. y = 9E 19x 0.0519 R = 0.9976 0 0.5 1 1.5 2 2.5 3 3.5 4 0 2E+18 4E+18 6E+18 Absorption Coefficient, N (cm 1 ) Number Density, N (cm 3 ) Absorption Coefficient vs. Number Density of Gly Absorption Coefficient vs. Number Density y = 1E 18x 0.2587 R = 0.9888 0 0.5 1 1.5 2 2.5 3 3.5 4 0.00E+00 2.00E+18 4.00E+18 Absorption Coefficient, N (cm 1 ) Number Density, N (cm 3 ) Absorption Coefficient vs. Number Density of Ala Absorption Coefficient vs. Number Density

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36 Figure 3 3. Plot of Absorption C oefficient vs. Number Density for Proline. The error bars account for all errors in the variables needed to derive this plot Figure 3 4 Plot of Absorption C oefficient vs. Number Density for Gly cine Gly cine The error bars account for all errors in the variables needed to derive this plot. y = 1E 18x + 0.0148 R = 0.9993 0 0.5 1 1.5 2 2.5 3 3.5 4 0 1E+18 2E+18 3E+18 Absorption Coefficient, N (cm 1 ) Number Density, N (cm 3 ) Absorption Coefficient vs. Number Density of Pro Absorption Coefficient vs. Number Density y = 3E 17x 0.6854 R = 0.998 0.3 1.3 2.3 3.3 4.3 5.3 6.3 1.00E+17 1.50E+17 2.00E+17 Absorption Coefficient, N (cm 1 ) Number Density, N (cm 3 ) Absorption Coefficient vs. Number Density of Gly Gly Absorption Coefficient vs. Number Density

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37 Figure 3 5 Plot of Absorption C oefficient vs. Number Density for Ala nine Gly cine The error bars account for all errors in the variables needed to derive this plot. Figure 3 6 Plot of Absorption C oefficient vs. Number Density for Gly cine Pro line The error bars account for all errors in the variables needed to derive this plot. y = 4E 17x 0.0649 R = 0.9899 0.3 1.3 2.3 3.3 4.3 5.3 6.3 1E+17 1.5E+17 2E+17 Absorption Coefficient, N (cm 1 ) Number Density, N (cm 3 ) Absorption coefficient vs. Number Density of Ala Gly Absorption Coefficient vs. Number Density y = 3E 17x + 0.1227 R = 0.9966 0.3 1.3 2.3 3.3 4.3 5.3 6.3 8E+16 1.3E+17 1.8E+17 Absorption Coefficient, N (cm 1 ) Number Density, N (cm 3 ) Absorption Coefficient vs. Number Density of Gly Pro Absorption Coefficient vs. Number Density

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38 Table 3 1 The Absorption Cross sections of the individual amino acids, their dipeptides, and the peptide bond in the dipeptides are recorded Sample unit (cm 2 ) peptide bond (cm 2 ) Ala 1.12E 18 N/A Gly 8.80 E 19 N/A Pro 1.03 E 18 N/A Gly Ala 3.65 E 17 3.45 E 17 Gly Gly 3.39 E 17 3.21 E 17 Gly Pro 2.54 E 17 2.35 E 17 Figure 3 7 Absorption data plotted as a function of the number of photons interacting with the Glycine solution at a beam energy of 1.00 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 7E 18x + 1.2344 R = 0.8005 0.5 0.7 0.9 1.1 1.3 1.5 1.7 0 2E+16 4E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.00 mJ/pulse Gly

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39 Figure 3 8 Absorption data plotted as a function of the number of photons interacting with the Glycine solu tion at a beam energy of 1.50 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 9 Absorption data pl otted as a function of the number of photons interacting with the Glycine solution at a beam energy of 2.00 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 4E 19x + 1.1507 R = 0.0132 0.5 0.7 0.9 1.1 1.3 1.5 1.7 0 5E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.50 mJ/pulse Gly y = 2E 18x + 1.042 R = 0.653 0.5 0.7 0.9 1.1 1.3 1.5 1.7 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n 2.00 mJ/pulse Gly

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40 Figure 3 10 Absorption data plotted as a function of the number of photons interacting with the Glycine solution at all beam energies. The error bars represent the standard deviation among the six samples tes ted at each beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 11 Absorption data plotted as a function of the number of photons interacting with the Alanine solution at a beam energy of 1.00 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 2E 18x + 1.0643 R = 0.1455 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n Absorption Coefficient vs. Number of Photons for Glycine Data from all Beam Energies y = 2E 18x + 1.3615 R = 0.1715 0.5 0.7 0.9 1.1 1.3 1.5 1.7 0 2E+16 4E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.00 mJ/pulse Ala

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41 Figure 3 12 Absorption data plotted as a function of the num ber of photons interacting with the Alanine solution at a beam energy of 1.50 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to genera te this plot. Figure 3 13 Absorption data plotted as a function of the number of photons interacting with the Alanine solution at a beam energy of 2.00 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 2E 18x + 1.6517 R = 0.5355 0.7 0.9 1.1 1.3 1.5 1.7 1.9 0 5E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.50 mJ/pulse Ala y = 2E 18x + 1.3357 R = 0.4374 0.5 0.7 0.9 1.1 1.3 1.5 1.7 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n 2.00 mJ/pulse Ala

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42 Figure 3 14 Absorption data plotted as a function of the number of photons interacting with the Alanine solution at all beam energies. The error bars represent the standard deviation among the six samples tested at each beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 15 Absorption data plotted as a function of the number of photons interacting with t he Proline solution at a beam energy of 1.00 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 2E 18x + 1.4996 R = 0.4237 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n Absorption Coefficient vs. Number of Photons for Alanine Data from all Beam Energies y = 1E 18x + 1.1334 R = 0.2834 0.5 0.7 0.9 1.1 1.3 1.5 1.7 0 2E+16 4E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.00 mJ/pulse Pro

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43 Figure 3 16 Abs orption data plotted as a function of the number of photons interacting with the Proline solution at a beam energy of 1.50 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 17 Absorption data plotted as a function of the number of photons interacting with the Proline solution at a beam energy of 2.00 mJ/pulse. The error bars represent the standard deviat ion among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 3E 19x + 1.0747 R = 0.005 0.5 0.7 0.9 1.1 1.3 1.5 1.7 0 5E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.50 mJ/pulse Pro y = 3E 19x + 0.9052 R = 0.0189 0.5 0.7 0.9 1.1 1.3 1.5 1.7 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n 2.00 mJ/pulse Pro

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44 Figure 3 18 Absorption data plotted as a function of the number of photons interacting with the Proline solution a t all beam energies. The error bars represent the standard deviation among the six samples tested at each beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 2E 19x + 0.9838 R = 0.0022 0.5 0.7 0.9 1.1 1.3 1.5 1.7 1.9 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n Absorption Coefficient vs. Number of Photons for Proline Data from all Beam Energies

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45 Figure 3 19 Absorption data plotted as a function of the number of photons interacting with the Glycine Glycine solution at a beam energy of 0.99 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all err ors of variables needed to generate this plot. Figure 3 20 Absorption data plotted as a function of the number of photons interacting with the Glycine Glycine solution at a beam energy of 1.19 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 6E 18x + 3.6804 R = 0.8874 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 0 2E+16 4E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n .99 mJ/pulse Gly-Gly y = 5E 18x + 3.5943 R = 0.9595 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 0 2E+16 4E+16 6E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.19 mJ/pulse Gly-Gly

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46 Figure 3 21 Absorption data plotted as a function of the number of photons interacting with the Glycine Glycine solution at a beam energy of 1.39 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 22 Absorption da ta plotted as a function of the number of photons interacting with the Glycine Glycine solution at a beam energy of 1.60 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account f or all errors of variables needed to generate this plot. y = 5E 18x + 3.4837 R = 0.7989 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 0 2E+16 4E+16 6E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.39 mJ/pulse Gly-Gly y = 6E 18x + 3.5321 R = 0.9179 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 0 5E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.60 mJ/pulse Gly-Gly

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47 Figure 3 23 Absorption data plotted as a function of the number of photons interacting with the Glycine Glycine solution at a beam energy of 1.87 mJ/pulse. The error bars represent the standard d eviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 24 Absorption data plotted as a function of the number of photons interacting with the Glycine Glyc ine solution at all beam energies. The error bars represent the standard deviation among the six samples tested at each beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 6E 18x + 3.5283 R = 0.8548 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.87 mJ/pulse Gly-Gly y = 6E 18x + 3.5995 R = 0.8764 2.8 2.9 3 3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n Absorption Coefficient vs. Number of Photons for Glycine Glycine Data from all Beam Energies

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48 Figure 3 25 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at a beam energy of 1.00 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all err ors of variables needed to generate this plot. Figure 3 26 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at a beam energy of 1.20 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 6E 18x + 3.182 R = 0.8536 2.4 2.6 2.8 3 3.2 3.4 3.6 0 2E+16 4E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.00 mJ/pulse Ala-Gly y = 5E 18x + 3.1434 R = 0.921 2.4 2.6 2.8 3 3.2 3.4 3.6 0 2E+16 4E+16 6E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.20 mJ/pulse Ala-Gly

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49 Figure 3 27 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine soluti on at a beam energy of 1.40 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 28 Absorption data plo tted as a function of the number of photons interacting with the Alanine Glycine solution at a beam energy of 1.60 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 7E 18x + 3.1497 R = 0.8685 2.4 2.6 2.8 3 3.2 3.4 3.6 0 5E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.40 mJ/pulse Ala-Gly y = 6E 18x + 3.3737 R = 0.9309 2.4 2.6 2.8 3 3.2 3.4 3.6 0 5E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.60 mJ/pulse Ala-Gly

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5 0 Figure 3 29 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at a beam energy of 1.80 mJ/pulse. The error bars represent the standard deviati on among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 30 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine so lution at a beam energy of 2.00 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 5E 18x + 3.3014 R = 0.8556 2.4 2.6 2.8 3 3.2 3.4 3.6 0 5E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.80 mJ/pulse Ala-Gly y = 6E 18x + 3.3015 R = 0.8142 2.4 2.6 2.8 3 3.2 3.4 3.6 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n 2.00 mJ/pulse Ala-Gly

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51 Figure 3 31 Absorption data plotted as a function of the number of photons interacting with the Alanine Glycine solution at all beam energies. The error bars represent the standard deviation among the six samples tested at each beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 32 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at a beam energy of 1.00 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 6E 18x + 3.2982 R = 0.8408 2.4 2.6 2.8 3 3.2 3.4 3.6 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n Absorption Coefficient vs. Number of Photons for Alanine Glycine Data from all Beam Energies y = 1E 17x + 2.0658 R = 0.8811 1 1.2 1.4 1.6 1.8 2 2.2 2.4 0 2E+16 4E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.00 mJ/pulse Gly-Pro

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52 Figure 3 33 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at a beam energy of 1.20 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 34 Absorption data plotted a s a function of the number of photons interacting with the Glycine Proline solution at a beam energy of 1.40 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all error s of variables needed to generate this plot. y = 1E 17x + 1.9298 R = 0.9834 1 1.2 1.4 1.6 1.8 2 2.2 2.4 0 2E+16 4E+16 6E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.20 mJ/pulse Gly-Pro y = 8E 18x + 2.0989 R = 0.949 1 1.2 1.4 1.6 1.8 2 2.2 2.4 0 5E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.40 mJ/pulse Gly-Pro

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53 Figure 3 35 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at a beam energy of 1.60 mJ/pulse. The error bars represent the standard deviation amo ng the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 36 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at a beam energy of 1.78 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 9E 18x + 2.0491 R = 0.9202 1 1.2 1.4 1.6 1.8 2 2.2 2.4 0 2E+16 4E+16 6E+16 8E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.60 mJ/pulse Gly-Pro y = 9E 18x + 1.8886 R = 0.9924 1 1.2 1.4 1.6 1.8 2 2.2 2.4 0 5E+16 Absorption Coefficient, N (cm 1 ) Number of Photons, n 1.78 mJ/pulse Gly-Pro

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54 Figure 3 37 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at a beam energy of 2.04 mJ/pulse. The error bars represent the standard deviation among the six samples tested at this beam energy and are assumed to account for all errors of variables needed to generate this plot. Figure 3 38 Absorption data plotted as a function of the number of photons interacting with the Glycine Proline solution at all beam energies. The error bars represent the standard deviation among the six samples tested at each beam energy and are assumed to account for all errors of variables needed to generate this plot. y = 1E 17x + 2.2841 R = 0.926 1 1.2 1.4 1.6 1.8 2 2.2 2.4 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n 2.04 mJ/pulse Gly-Pro y = 9E 18x + 2.1025 R = 0.9371 1 1.2 1.4 1.6 1.8 2 2.2 2.4 0 5E+16 1E+17 Absorption Coefficient, N (cm 1 ) Number of Photons, n Absorption Coefficient vs. Number of Photons for Glycine Proline Data from all Beam Energies

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55 Table 3 2 The s lopes of the Absorption Coefficient vs. Number of Photons plots for Glycine Glycine are recorded in the left co lumns for each beam energy These values were averaged and the standard deviations were calculated. The slopes of the plot that include s all the beam energies are recorded in the last column. This value relates well calculations. Gly Gly 0.99 mJ/pulse 1.19 mJ/pulse 1.39 mJ/pulse 1.60 mJ/pulse 1.87 mJ/pulse Average Std. Dev. All Energies Slope 6.20E 18 5.13E 18 5.06E 18 5.82E 18 5.56E 18 5.56E 18 4.79E 19 5.53E 18 Table 3 3. The slopes of the Absorption Coefficient vs. Number of Photons plots for Glycine Proline are recorded in the left columns for each beam energy. These values were averaged and the standard deviations were calculated. The slopes of the plot that includes all the beam energies are recorded in the last column. This value relates well calculations. Gly Pro 1.00 mJ/pulse 1.20 mJ/pulse 1.40 mJ/pulse 1.60 mJ/pulse 1.78 mJ/pulse 2.04 mJ/puls e Average Std. Dev. All Energies Slope 9.74E 18 9.85E 18 7.84E 18 8.99E 18 9.29E 18 9.79E 18 9.25E 18 7.66E 19 9.31E 18 Table 3 4. The slopes of the Absorption Coefficient vs. Number of Photons plots for Alanine Glycine are recorded in the left columns for each beam energy. These values were averaged and the standard deviations were calculated. The slopes of the plot that includes all the beam energies are recorded in the last column. This value relates well calculations. Ala Gly 1.00 mJ/pulse 1.20 mJ/pulse 1.40 mJ/pulse 1.60 mJ/pulse 1.80 mJ/pulse 2.00 mJ/pulse Average Std. Dev. All Energies Slope 5.65E 18 5.21E 18 7.21E 18 5.68E 18 4.96E 18 5.91E 18 5.77E 18 7.86E 19 5.71E 18

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56 Figure 3 39 Representative plot of Absorption Coefficient vs. Number of Photons. Plot was used to understand the relation between the number of broken peptide bonds and the number of photons. Table 3 5 The number of photons it takes to break one peptide bond, for each of the dipeptides, is reported in the fifth column. Dipeptide PB m # Broken Bonds per # Photons # Photons to Break One Bond Gly Gly 3.2 0 E 17 5.53 E 18 0.172 8 5. 79 Ala Gly 3.45 E 17 5.71 E 18 0.1658 6.03 Gly Pro 2.35 E 17 9.31 E 18 0.3966 2.52

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57 CHAPTER 4 ANALYSIS AND DISCUSS ION Non Perturbation Experiments First, the results to the first part of this experiment will be discussed. As was mentioned in the Materials and Methods c hapter, the purpose of this section of the experiment was to determine the absorption cross sections of the amino acids and the peptide bonds. P lots of Absorption Coefficient vs. Number Density were used to der ive this information. Figures 3 1 3 6 are the plots obtained for Glycine, Alanine, Proline, Glycine Glycine, Alanine Glycine, and Glycine Proline, respectively. All of the plots display a very linear trend; the smallest R value among them is 0.9888. All of the plots suggest that as one increa se s the number density (the number of absorbers) the absorption coefficient increases (more energy is absorbed) which physically makes sense. The slopes of these plots represent the absorption cross sections of the amino acids and their dipeptides. These absorption cross sections are tabulated in T able 3 1 The absorption cross sections of the amino acids range from 8.8E 19 cm 2 to 1.1E 18 cm 2 whereas the absorption cross sections of the dipeptides range from 2.5E 17 cm 2 to 3.6E 17 cm 2 ; a full order of mag nitude greater than the individual amino acids. Again, this is attributed to the peptide bond that is present in the dipeptides. The absorptions coefficients of the amino acids, their dipeptides, and the peptide bond are related to one another by E quation 2 4; however, E quation 2 5 represents this relationship with just absorption cross sections assuming the number of dipeptide molecules is equal to the number of peptide bonds and equal to one half the number of indi vidual amino acids. Therefore, E quations 2 6 2 8 were generated and the absorption cross sections of the peptide bonds, for each of the dipeptides, were calculated and tabulated in T able 3 1 From these values, it was calculated that the peptide bond was responsible for roughly 93% of the absor ption by these dipeptides, which relates well to Fisher

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58 in collagen (2004). Perturbation Experiments For this part of the research, the perturbation laser beam w as used and a relationship between absorption coefficient and the number of photons experienced by the sample solution was desired. With this information one could derive a relationship between the peptide bonds and the photons delivered by the 193 nm exc imer laser. First, it is useful to examine the amino acid resu lts which refer to F igures 3 7 3 18 As mentioned in the Methods chapter, the amino acids were tested at three different beam energies: 1.00 mJ/pulse, 1.50 mJ/pulse, and 2.00 mJ/pulse. The re sults for Gl ycine are pictured in F igures 3 7 3 9 All of these graphs depict a small negatively sloped trend, which suggest that as the number of photons increases the amount of energy absorbed decreases, leading one to conclude that absorbers are dest royed in this process. It is assumed that all three slopes, from the three beam energies, should be equivalent because the absorption coefficient should decrease linearly as the number of photons increases. However, the data from these graphs are very erra tic, with R values ranging from 0.013 to 0.801. Due to this, a plot with all data points on it was created (i.e. all th ree beam energy data) (Figure 3 10 ). From this graph a negatively sloped trend was derived ; however, one can fit a linear line to any set of data to derive a general trend, while the R value is what makes the trend significant or not. For this data an R value of 0. 146 was derived suggesting that this data is unreliable to make clear the trend Due to the non linearity of the data, it i s concluded that changing the n umber of photons incident on a G lycine solution does no t change the absorption coefficient of that solution a significant amount. The same result was found for Alanine and P rolin e solutions; refer to F igures 3 11 3 14 and F igures 3 15 3 18

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59 respectively. Overall, the perturbation of the cross section of isolated amino acids by the excimer laser is negligible over these ranges. Next, the dipeptides we re analyzed; refer to F igures 3 19 3 38 The Glycine Glycine solution was tested at five beam energies: 0.99 mJ/pulse, 1.19 mJ/pulse, 1.39 mJ/pulse, 1.60 mJ/pulse, and 1.87 mJ/pulse. The absorption coefficient was plotted against the number of photons incident on the solution for each beam ener gy (Figures 3 19 3 23 ). Linear lines were fit onto this data and the slopes from thes e lines were recorded in T ables 3 2 3 4 (Note: the absolute values of the slopes are reported ). The slopes are generally consistent, with a standard deviation of 4.79 e 19 and an average of 5.56e 18 cm 1 /number of photons. As with the amino acids, all of the data from all beam energies was summarized onto one plot. In compiling all of the data, each set was normalize d to a common initial absorption coefficient which ac counts for slight differences (i.e. vertical offset) in the single energy experiments due to sample to sample variations in absolute baseline absorbance. It is noted, however, that the individual single energy experiments were all characterized by very sim ilar slope. The R value found for Glycine is significantly better than those f ound for the amino acids (0.0022, 0.1455, and 0.4237 ); therefore, it is conclude d that for Glycine Glycine, the number of photons seen by the dipeptide is linearly related to the absorption coefficient of that dipeptide. The same analysis was implemented for the Alanine Glycine and Glycine Proline dipeptides, which were tested at six different beam energies, and similar results were found (Table s 3 3 and 3 4) An R value of 0.8408 was found for the Alanine Glycine dipeptide and 0.9371 was found for the Glycine Proline dipeptide. As T able s 3 2 3 4 show there is very good agreement between the slope calculated from the

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60 plots are used for future calculations. Speaking of future calculations, the next objective was to derive a relationship between the n umber of broken peptide bonds and the number of photons acting on the peptide bonds. Figure 3 39. Representative plot of Absorption Coefficient vs. Number of Photons. Plot was used to understand the relation between the number of broken peptide bonds a nd the number of photons. To understand the relation between the number of peptide bonds bro ken and the number of photons, F igure 3 39 (depicted above for easier reference ) was needed. This figure represents a plot of absorption coefficient vs. number of P is the change in the number DP is the change in the number of dipeptides or the change in the number of broken peptide bonds, N DP DP is the absorption coefficient of the dipeptides at point 1, and (N DP DP DP + DP AA DP AA are the absorption cross sections of the dipeptides and amino acids, respectively). When a peptide bond is broken, and after reacting with water, two amino acids are left over, an d this is the reason DP AA term. The following relations are defined : (4 1) (4 2)

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61 Equation 4 2 needs to be expanded and simplified: (4 3) In this equation, m ( ) is th e slope of the plot in F igure 3 39 which is negative, and m (+) is the absolute value, as it is recorded in T able s 3 2 3 4 Equation 4 3 shows that one can calculate the number of bonds broken per photon by simply dividing the slope derived from the second part of this experiment (Table s 3 2 3 4 ) by th e peptide bond cross section calculated from the first part of this experiment (Table 3 1 ). By inverting this value one can also derive how many photons it takes to break one peptide bond. These resul ts are tabulated in T able 3 5 The Glycine Glycine and Alan ine Glycine dipeptides took 5.8 and 6.0 photons to break one peptide bond, respectively. The Glycine Proline di peptide, however, only took 2.5 photons to break its peptide bond. T his difference is attributed to the difference in the molecular structure of the amino acids. Figure 2 5 shows the molecular structures of these amino acids. Glycine Glycine and Alanine Glycine have the same general structure as one another; whereas, Glycine Proline include s a adjacent to the peptide bond This cyclic structure must enhance the photon peptide bond interactions since it takes less photons of average energy to break the bond when compared to the peptide bonds of Glycine Glycine and Glycine Proline.

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62 CHAPTER 5 CONCLUSIONS AND FUTU RE WORK In this study, the fundamental building blocks of collagen, namely amino acids, were explored in the context of 193 nm laser interactions at the sub ablative level. First, the absorption cross sections of the isolated amino acids (Glycine, Alanine, and Pro line) and the dipeptides ( Gly Gly, Ala Gly, and Gly Pro ) were measured from solutions. Knowing the cross sections, the influence of the 193 nm radiation on the various cross sections and peptide bonds was accessed. It was found that the perturbation of the cross section s of isolated amino acids by the excimer laser had a negligible effect on the absorption coefficient of these amino acids for this range of energies. The opposite can be said about the dipeptides. As the number of photons interacting with the dipeptides increased the absorption coefficient of the dipeptides decreased suggesting that peptide bonds (the main absorbers of these photons at 193 nm) were directly broken. A relationship between the number of photons and the number of broken bonds w as then formulated and the number of photons it took to break one peptide bond in Glycine Glycine, Alanine Glyci ne, and Glycine Proline was 5.8, 6.0, and 2.5 respectively. Glycine Glycine and Alanine Glycine displayed similar results, whereas Glycine Pro line needed half the photons to break one of its peptide bonds. This difference was attributed to the difference in the molecular structure of the dipeptides, with Glycine Proline including a cyclic ring structure that was in close proximity to the peptide bond. For future work, it is recommended that more complex amino acids and dipeptides which represent collagen tissue and other more complicated tissue structures be studied. Tripeptides, such as Gly Pro Hyp, are one example, and they can be studied to determine if the peptide bonds within this molecule break simultaneously or at different times The molecule Proline Glycine could also be tested to determine if it produces results similar to Glycine Glycine and Alanine

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63 Glycine or to Glycine Proline. The difference between Glycine Proline and Proline Glycine is the proximity of the cyclic ring structure in Proline to the peptide bond. Ultimately, a better understanding of the laser tissue interactions at sub ablative values can lead to a more detailed expl anation of laser tissue ablation as well as help in the design of advanced optical biopsy methods based on deep UV laser tissue interactions.

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64 APPENDIX ERROR ANALYSIS A simple error calculation was implemented for various parameters using the fol lowing formula: (A 1) This formula was used to calculate the error s of the number d ensities (N), the number of p ho tons (n), the absorption c oefficients ( N), and the n umber o f photons it takes to break one peptide b ond (k). Fo r the number d ensity (Equation 2 3 the molar mass were neglected. The error for the density was determined by the error of the scale, which was ~1 mg; therefore, the error of our density was ~1 mg/200 mL H 2 O for dipeptides and ~1mg/48 mL H 2 O for amino acids. This erro r led to a 12 20% error in the number d ensity; however, for future calculations it is assumed this error was accounted for by the standard deviation of the t at each concentrati on. For the number of p hotons (E quation 2 9), the errors caused by the number of pulses and the photon energy were neglected. Therefore, the error of the number of photon s was determined by the error of the photon energy per pulse. During the experiment, an error of 0.05 mJ/pulse was recorded which led to a 5% error in the number of photons. Again, this error was assumed to be accounted for by the standard deviation of th e absorption coefficient beam energy. The error of the absorption c oefficient was calculated in two ways. First, the non perturbation experiments took use of E quation A 1and E quation 2 1 ( ) w by the three samples tested for each concentration The error bars in F igures 3 1 3 6 represent

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65 this er ror. Secondly, the perturbation experiments simply took the error of the absorption coefficient as the standard deviation of the absorption coefficient determined by the six samples tested for each beam ener gy. The error bars in F igures 3 7 3 38 illustra te this error. In order to determine the errors of the slopes derived in F igures 3 1 3 38, a Monte Carlo error analysis was conducted. For each point on these graphs, a random number from a Gaussian distribution with the mean and standard deviation of th at poi nt was computed. A new slope wa s then calculated from this set of new data points. This process wa s repeated five thousand times and then the average and standard deviation of the slopes of these graphs we re calculated from these trials. This error a nalysis pr ovided the errors in the slopes representing the absorption cross sections of the amino acids and dipeptides, as well as the error of the slope (m) of the absorption coefficient vs. number of photons. Using these errors, the error s of the absorpt ion cross section s of the peptide bonds for each dipeptide were calculated utilizing E quation A 1. Based on th e e rrors of the absorption cross sections of the peptide bonds, the error of the slope (m) of the absorption coefficient vs. number of photons and utilizing E quation A 1 the error in the number of photons it takes to break one peptide bond for each dipeptide was derived. Glyci ne Glycine has an error of 1.48 (number of photons it takes to break one peptide bond), Alani ne Glycine has an error o f 1.20 and Glycine Proline has a n error of 0.48 This error in the number of photons it takes t o break one peptide bond is significant to the average values we recorded in T able 3 5 (~19 26 % error). This error derives from the variation of the laser energy from pulse to pulse. Another source of error could have been caused by bubbles that formed in about 10% of the sample cells. These bubbles could easily distort the optical properties of the solutions that were made and, therefore, cause erratic resu lts. Typical laser beam fluctuations are summarized in T able A 1, as measured with the Ophir power meter.

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66 Table A 1 This table records the beam energy fluctuation data for the 193 nm excimer laser used. 100 pulses of the beam were used to obtain this dat a. Average (mJ) Minimum (mJ) Maximum (mJ) Std. Dev. (mJ) 1.42 1.32 1.49 0.034

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67 LIST OF REFERENCES Fisher B.T., and Hahn D. W. (2004). Measurement of small signal absorption coefficient and absorption cross section of collagen for 193 nm excimer laser light and the role of collagen in tissue ablation, Appl. Optics 43:5443 5451 Fisher, B.T., and Hahn D. W. (2007). Development and numerica l solution of a mechanistic model for corneal tissue ablation wit h the 193 nm ArF excimer laser, J. Opt. Soc. Am. 24 : 265 277. Fisher, B.T., and Hahn D. W. (2011). Real time measurement of ArF excimer laser corneal tissue ablation rates using cross correlation of laser waveforms, Optics Exp. 19 : 4231 4241 Gorner, H. (1994). Photochemistry of DNA and related biomolecules: quantum yields and consequences of photoioniza tion, J. Photochem. Photobiol. 26 : 117 139. Jimnez, J. R. Rodriguez Marn, F. Anera, R. G. and Jimnez del Barco, L. (2006). Deviations of Lambert Opt. Express 14 : 5411 5417. Kitai M. S. Popkov, V. L. Semchishen, V. A. and Kharizov, A. A. (1991). The Physics of UV Laser Cornea Ablation, IEEE J. Quant. Elect. 27 : 302 307. Krueger R. R. and Trokel, S. L. (1985). Quantization of corneal ablation by ultraviolet laser light, Arch. Ophthalmol. 103 : 1741 1742. B. Bityurin, N. Anisimov, S. Arnold, N. and Buerle, D. (1996). The role of excited species in ultraviolet laser materials ablation III. Non stationary ablation of organic polymers, Appl. Phys. 62 : 397 401. Nikogo syan D. N. and Gorner, H. (1995). Photolysis (193 nm) of aliphatic amino acids in aqueous solution, J. Photochem. Photobiol. 30 : 189 193. Nikogosyan D. N., and Gorner, H. (1998). Towards the laser photochemistry of the cornea: studies of the most common and highly absorbing aliphatic amino acids in collagen, J. Photochem. Photobiol. 47 : 63 67. Nikogosyan D. N., and Gorner, H. (1999). Laser Induced Photodecomposition of Amino Acids and Peptides: Extrapolation to Corneal Collagen, IEEE J. Sel. Top. Quant. 5 : 1107 1115. Paltauf G., and Dyer, P. E. (2003). Photomechanical Processes and Effects in Ablation, Chem. Rev. 103 : 487 518. Razhev, A. M. Zhupikov, A. A. and Churkin, D. S. (2009). Investigating the action of the 193 nm and 223 nm radiation of excimer lasers on the cornea of the human eye in refractive surgery, J. Opt. Technol. 76: 263 267

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68 Shanyfelt, L. M. (2008). ArF excimer laser corneal ablation: effects of laser repetition rate a nd fundamental laser tissue coupling, PhD Dissertation, University of Florida. Smith, S. E. Shanyfelt, L. M. Buchanan, K. D. and Hahn, D. W. (2011). Differential laser induced perturbation spectroscopy using a deep UV excimer laser, Optics Letters 36: 21 16 2118 Srinivasan, R. Braren, B. Seeger, D. E. and Dreyfus, R. W. (1986). Photochemical Cleavage of a Polymeric Solid: Details of the Ultraviolet Laser Ablation of Poly(methyl methacrylate) at 193 and 248 nm, Macromolecules 19 : 916 921. Sutcliffe E. and Srinivasan, R. (1986). Dynamics of UV laser ablation of organic polymer surfaces, J. Appl. Phys. 60 :3315 3322 Tokarev, V. N. Lunney, J. G. Marine, W. and Sentis, M. (1995). Analytical thermal model of ultraviolet laser ablation with single photon absorption in the plume, J. Appl. Phys. 78 : 1241 1246. Vogel A., and Venugopalan, V. (2003). Mechanisms of Pulsed Laser Ablation of Biological Tissues, Chem. Rev. 103 : 577 644.

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69 BIOGRAPHICAL SKETCH Kristofer Howell Loper was born in Tampa, Florida, i n 1987, the youngest child of Norman and Lorraine Loper Kristofer was raised and went to high school in Tampa. In August 2005, he began his undergraduate studies at the University of Florida. Kristofer graduated with a Bachelor of Science degree in aerosp ace engineering with cum laude h onors and a Bachelor of Science degree in mechanical engineering with summa cum laude h onors in May 2010. He decided to continue his studies in graduate school at the University of Florida. This work is the culmination of hi s pursuit of a Master of Science degree in mechanical e ngineering.