Group Title: 7th International Conference on Multiphase Flow - ICMF 2010 Proceedings
Title: 10.3.1 - Novel techniques for imaging particle in human airway model using near-infrared spectrum
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Title: 10.3.1 - Novel techniques for imaging particle in human airway model using near-infrared spectrum Experimental Methods for Multiphase Flows
Series Title: 7th International Conference on Multiphase Flow - ICMF 2010 Proceedings
Physical Description: Conference Papers
Creator: Bunchatheeravate, P.
Curtis, J.S.
Brown, S.
Marijnissen, J.
Publisher: International Conference on Multiphase Flow (ICMF)
Publication Date: June 4, 2010
 Subjects
Subject: lung deposition
near-infrared
electrospray
 Notes
Abstract: Deposition of micron size particles in the human airway tree presents a critical issue in pharmaceutics and toxicology. To understand the behavior of the inhaled particles, many experimental studies and CFD simulations have been conducted. Experimental studies involve determining the total and regional deposition in the human airway on human volunteers, animals, and airway models. Total deposition can be easily determined using the concentration difference between the inhalant and the exhalent for live subjects or using weight differences for airway models. Regional deposition, on the other hand, is more troublesome. Radio-labeled particles are the main instrument in visualizing the regional deposition in both live and model experiments. Health issues and numerous precautions are issues for both the investigator and the subject, leading to difficulties in experimentation. In the present work, a novel technique for imaging and quantification of regional deposition in the human airway is developed. This method makes use of safe and biodegradable Poly(lactic-co-glycolic acid) (PLGA) particles that are loaded with Near-Infrared (NIR) dye. The NIR fluorescence offers a nondestructive analysis of the airway. Some initial studies applying this technique are presented. NIR-loaded particles are generated at a constant rate via electrospraying. The particles are pulled through a glass model of the human airway tree. The deposited particles are imaged with the IVIS imaging system (Xenogen). Results indicate that the NIR dye offers a way to visualize the particle deposition pattern with high resolution and good reproducibility. Furthermore, the deposition fraction in each region of the model can be quantified using the amount of photons generated from the dye. With this imaging technique, particle deposition in the human airway, as well as other complex geometries, can be easily visualized.
General Note: The International Conference on Multiphase Flow (ICMF) first was held in Tsukuba, Japan in 1991 and the second ICMF took place in Kyoto, Japan in 1995. During this conference, it was decided to establish an International Governing Board which oversees the major aspects of the conference and makes decisions about future conference locations. Due to the great importance of the field, it was furthermore decided to hold the conference every three years successively in Asia including Australia, Europe including Africa, Russia and the Near East and America. Hence, ICMF 1998 was held in Lyon, France, ICMF 2001 in New Orleans, USA, ICMF 2004 in Yokohama, Japan, and ICMF 2007 in Leipzig, Germany. ICMF-2010 is devoted to all aspects of Multiphase Flow. Researchers from all over the world gathered in order to introduce their recent advances in the field and thereby promote the exchange of new ideas, results and techniques. The conference is a key event in Multiphase Flow and supports the advancement of science in this very important field. The major research topics relevant for the conference are as follows: Bio-Fluid Dynamics; Boiling; Bubbly Flows; Cavitation; Colloidal and Suspension Dynamics; Collision, Agglomeration and Breakup; Computational Techniques for Multiphase Flows; Droplet Flows; Environmental and Geophysical Flows; Experimental Methods for Multiphase Flows; Fluidized and Circulating Fluidized Beds; Fluid Structure Interactions; Granular Media; Industrial Applications; Instabilities; Interfacial Flows; Micro and Nano-Scale Multiphase Flows; Microgravity in Two-Phase Flow; Multiphase Flows with Heat and Mass Transfer; Non-Newtonian Multiphase Flows; Particle-Laden Flows; Particle, Bubble and Drop Dynamics; Reactive Multiphase Flows
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Resource Identifier: 1031-Bunchatheeravate-ICMF2010.pdf

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7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


Novel Techniques for Imaging Particles in a
Human Airway Model using Near-Infrared Spectrum


Poom Bunchatheeravatea, Jennifer S. Curtisa, Scott Brownb, Jan Marijnissenc


aChemical Engineering Department, University of Florida, Bldg. 723, Gainesville, FL 32611, USA
pbuncha@tufl.edu

bParticle Engineering Research Center, University of Florida, Particle Science & Technology, Gainesville, FL 32611, USA

CDepartment of Chemical Technology, Delft University of Technology, Julianalaan 136, 2628 BL Delft, The Netherlands

Keywords: Lung Deposition, Near-Infrared, Electrospray



Abstract

Deposition of micron size particles in the human airway tree presents a critical issue in pharmaceutics and toxicology. To
understand the behavior of the inhaled particles, many experimental studies and CFD simulations have been conducted.
Experimental studies involve determining the total and regional deposition in the human airway on human volunteers, animals,
and airway models. Total deposition can be easily determined using the concentration difference between the inhalant and
the exhalent for live subjects or using weight differences for airway models. Regional deposition, on the other hand, is more
troublesome. Radio-labeled particles are the main instrument in visualizing the regional deposition in both live and model
experiments. Health issues and numerous precautions are issues for both the investigator and the subject, leading to
difficulties in experimentation.

In the present work, a novel technique for imaging and quantification of regional deposition in the human airway is developed.
This method makes use of safe and biodegradable Poly(lactic-co-glycolic acid) (PLGA) particles that are loaded with
Near-Infrared (NIR) dye. The NIR fluorescence offers a nondestructive analysis of the airway. Some initial studies
applying this technique are presented. NIR-loaded particles are generated at a constant rate via electrospraying. The
particles are pulled through a glass model of the human airway tree. The deposited particles are imaged with the IVIS
imaging system (Xenogen). Results indicate that the NIR dye offers a way to visualize the particle deposition pattern with
high resolution and good reproducibility. Furthermore, the deposition fraction in each region of the model can be quantified
using the amount of photons generated from the dye. With this imaging technique, particle deposition in the human airway,
as well as other complex geometries, can be easily visualized.


Introduction

Particle deposition in the lung has always been an area
of interest. Whether the study is used for toxicology
purposes (Kreyling et. al., 2004) or pharmaceutical purposes
(Brand et. al., 2005), much work and effort has been put
into determining where the particles will deposit. For a
long time, particle deposition experiments simply compared
the difference between the inhaled and exhaled particle
concentration (Chalupa et. al., 2004). It was not until
medical imaging devices became available that regional
deposition was imaged. These devices can show different
regions of deposition, enabling the ability to visualize the
deposition.

MRI, CT, and SPET/PET scans are the main devices
used in visualizing the regional deposition. CT and PET
scans can yield good deposition results but there are risks
involved in both techniques. CT scan shines x-rays
through the chest, into the lung, and x-rays are absorbed by
both the particles and tissues. In addition, safe and


inhalable particles that show strong contrast with CT scans
are very limited. Like the CT scan, the SPET/PET scan
can yield high quality images of the deposition (Eberl et. al.,
2001). But PET scans require use of radioactive particles;
the most commonly radiotracer used is 99m-Technetium
(Dolovich and Labiris, 2004). Exposure to harmful
substances is generally kept to a minimum, but prolong or
repeated exposure may increase the risk of cancer and other
side effects. MRI, on the other hand, uses magnetic
resonance and magnetic particles for imaging. MRI is
deemed safe, but the resolution is poor compared to other
techniques. The current study investigates the use of
fluorescence dye to image particle deposition in the human
lung. Fluorescence dye has been used previously to study
particle deposition in model and animal lungs. However,
fluorescence dye has not been used in a non-invasive
imaging study. The current study seeks to look at a
specific type of dye, the near infrared (NIR) dye.

NIR-based technologies have been around since the
1960s. The NIR spectrum consists of wavelength in the






7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


region between visible light and inferred light. This region
contains light with wavelengths longer than that of visible
light, between 780 to 2500 nm. The NIR spectrum is
found to have numerous advantages in medical imaging
(Mark and Campbell, 2008). In term of operation, NIR
can provide quick results with good accuracy, allowing for
quick sample turn over. In term of safety, the NIR
spectrum is noninvasive and does not require the use of
toxic or corrosive chemicals. This makes the NIR
spectrum ideal for use in noninvasive in-vivo imaging. In
terms of cost, NIR does require high initial cost for the
imaging machine but the cost for imaging individual
samples is rather low compared to other techniques. In the
medical and pharmaceutical industry, NIR spectrum
imaging is a safe and cost effective way to analyze chemical
composition in drug pellets and in-vivo imaging in animals.
NIR dye can also be a safe and cost effective way to
conduct deposition experiments in both humans and
animals.


Experimental Facility

Experimental Setup

The lung model used is based on the first 4 generations
of Weibel's Model A (Weibel 1963); this includes the
airways from the trachea to the 3rd branching. Model A is
the simplest structure for modeling the human airway; it is
ideal for testing new techniques. Weibel only specifies the
airway dimension and the branching angle. Hence,
construction base on this alone would be a flat 2D model.
Tawhai et. al. (2000) indicated that the branching plane of
the lung is, on average, at an angle of 900; this angle is
included in the present model. The lung model is made
from borosilicate glass by a glass blower (ACE Glass).
For the deposition experiments, the model is coated with a
thin layer of silicone oil. The purpose of the coating of
silicone oil is two-fold. First, the silicone oil helps to
simulate mucus lining in the lung; it helps to capture
particles and reduce particle bouncing. Second, the
conductivity of the silicone oil is used to dampen out any
surface charge occurring in the borosilicate glass.

Table 1: Dimensions of Weibel's Model A


Generation


Branch


Diameter
(cm)


Length
(cm)


Cross-sectio
nArea(cmr)


GO 1 1.8 12 2.54 30.5
G1 2 1.22 2.33 2.33 11.25
G2 4 0.83 2.13 2.13 3.97
G3 8 0.56 2 2 1.52

The setup for the deposition experiments is shown in
Figure 1. This setup consists of two parts the particle
generation section and the deposition section. The particle
generation is done using electrospray. The electrospray is
set up at the top of the unit and particles move downward
from the top. The vertical set up helps to reduce particle
loss due to settling and wall loss. The deposition section
consists of a spacer section, the lung model, and the pump.
Open space is provided between the spraying nozzle and the
lung allowing sufficient time for particle formation and for


Filter Vacuum
Pump
Figure 1: Schematic of the spraying and deposition set up

the flow to develop. The lung model is connected to the end
of the spacer section. The model is positioned vertically,
the same as humans in the upright position. A filter is
placed after the glass model to collect any particles
uncollected by the glass model. A vacuum pump is
connected to the lung model to generate flow.

Particle Generation

Particles used in these studies are made from
biodegradable polymer poly(lactic-co-glycolic) acid
(PLGA). Solid PLGA particles are generated by
electrospray. Electrospray is chosen for this application
because of its ability to generate monodispersed aerosol and
produce a constant particle flux over time. A polymer
solution is made by mixing 85:15 PLGA (DURECT Corp.)
in a solution of dichloromethane and acetone. An NIR dye,
3,3'-diethylthiatricarbocyanina iodide (Sigma-Aldrich), is
added into the solution. The purpose of the addition of the
dye is two fold: first, to coat the particles with the dye;
second, to increase the conductivity of the solution and help
the spraying process. The shape and size of the particles
generated by electrospray are strongly dependent on the
properties of the spraying solution. The spraying solution
used in this study was designed to generate solid spherical
particles with size range between 1 to 3 microns. The
physical properties of the spraying solution are listed in






7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


Table 2.


.*


10orn


Figure 2: Sample of the particles generated through electrospray

The spraying solution is fed through a nozzle. A high
voltage source is connected to the nozzle to supply electrical
charge to the solution exiting the nozzle. A highly charged
ring is place underneath the nozzle to induce a potential
difference across the nozzle and the ring. The potential
difference applied to the spraying solution generates a cone
at the nozzle tip and atomizes liquid into a fine spray. The
dichloromethane-acetone solution in each droplet will
evaporate, leaving behind solid PLGA particles embedded
with NIR dye. The evaporation process happens rapidly,
thus forming the solid particles before reaching the lung
model. The spacer section in the set up is used to ensure
that all the solution evaporates.

Table 2: Properties of the spraying solution

Polymer Concentration 0.75%
by weight
Density 1160kg/m3
Conductivity 0.00002S/m
Surface Tension 0.0274N/m
Viscosity 0.00063Pa-s

Highly charged particles are generated from
electrospray. To neutralize the particles, sharp needles
connecting to a high voltage source were introduced into the
system. A total of 4 needles were used. The needles
were soldered onto a copper ring with the sharp end of the
needle pointing toward the center. A high voltage, with a
field opposite to the ring and nozzle, is applied to the
needles. The sharp end of the needle creates a corona
discharge that neutralized the particles flowing through the
ring.

A sample of generated particles is collected on a
microscopic slide. The sample is viewed under the
microscope to inspect particle size and morphology.
Particle size distribution can be determined using the
particle analysis algorithm in ImageJ. Statistics of the
particles are calculated based on the projected area diameter.


Deposition and Imaging

Once generated, particles are directed into the lung
model. The flow in performed with a vacuum pump; the
vacuum is used to simulate the inhalation flow. The flow
rate used is 30LPM; this flow rate is equivalent to that of an
average adult male at rest. To ensure a very strong signal,
the deposition was carried out over a period of 4 hours.

Deposition in the lung model is imaged with the IVIS
medical imaging system (Xenogen). The NIR dye is found
to have a good signal using an excitation wavelength of
710nm, an emission wavelength of 780nm, and an exposure
time of 2.5s. Multiple pictures from a single experimental
trial were taken from the IVIS to visualize deposition at
each region of the model. The IVIS uses an
auto-calibration system so a control is needed for
comparison between pictures. Calibration tubes were used
as a medium for comparing individual pictures. Each
calibration tube contains a known amount of polymer and
dye. The calibration tubes are present in all the pictures
taken and are used to create a calibration curve that relates
the total photon flux in an area to the mass of polymer
present. The calibration tubes contain 0, 1, 2, 4, and 5pm
of the spraying solution. Three different sets of identical
tubes were made to account for any variations in the signal.

Results and Discussion

Some examples of particles images taken from the
optical microscope are shown in Figure 2. The particles in
the pictures are spherical in shape and are of uniform size
distribution. The pictures confirm the use of electrospray
to generate monodispersed particles. The uniform particle
flux is confirmed by monitoring the spray over time. To
ensure a statistically significant particle size distribution,
multiple pictures from the optical microscope were used to
analyze the particle size distribution. A histogram of the
particles is shown in Figure 3 and the particle statistics are
shown in Table 3. The standard deviation and the
histogram show a narrow size distribution.


* I


*
*


* *o
10 "
*


*



. *



* *


*










120.00%

100.00%

80.00%

60.00%

40.00%

20.00%

0.00%


o t r^ \ N M N \~ N \0~tCZ I M O C .0 d
Bin
Figure 3: Histogram of particles




Multiple pictures are taken from the IVIS from each
individual trial. Each picture includes the calibration tubes
for a specific region of the lung model. A picture of the
calibration tubes is shown in Figure 4 (left). The total
photon flux emitted from each tube is used to make a
calibration curve, Figure 4 (right). The calibration curve
shows a linear relationship between the total photon flux
produced from each calibration tube to the amount of
polymer associated with each tube. The general
relationship between the photon flux and polymer mass is
given by the linear relation:

Flux = 5.149 x 1012(Polymer)+ 2.153107

The intercept for the linear relation indicates that there are
photons generated from the background, and the slope
represents the relation between the flux and the polymer
mass. The background signal is due to the reflection of
light off the background and the curvature of the tube.

A specific calibration curve is made for each picture. The
photon flux-polymer mass relation in each picture exhibits a
slightly different slope and intercept the variations are due
to the effects of light interaction and the auto-calibration of


II




tc'
(4
84


7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010

Table 3: Statistical property of the particle

Projected Area Diameter (pm)


Geometric Mean 2.37

Geometric Standard 1.18
Deviation

Median 2.35

Mode 2.40


the IVIS system. Both the slope and intercept are generally
within the same magnitude with about 3% deviation for the
slope and 5% deviation for the intercept. Since there is
little deviation from the calibration curve, the curve can be
used to normalize each of the separate pictures. The
day-to-day variation of the calibration curve is 4.92% for
the slope and 17.95% for the interception. The relation is
similar but the background noise changes from day to day.

A compilation picture of a single trial is presented in Figure
5. Figure 5 also outlines each region of deposition that
will be examined. Using the photon flux-polymer mass
relationship, the photon flux in each region of the lung
model is converted into its mass value. The amount of
deposition found in each region is normalized with the total
mass to get the percentage of total deposition in the lung
model. The actual mass calculated with the relationship
was not used as a comparison; day-to-day conditions change
the amount of particles collected on the walls of the
electrospray chamber. The sheath flow of air was used to
counter this effect, but the effects of the sheath flow
diminish significantly during the neutralization of the
particles. The regional deposition values from 3 different
deposition trials, average, and standard deviation are shown
in Table 4.


3.00E+08


2.50E+08
C
S2.00E+08

5 1.50E+08
C-
1.00E+08
I-
5.00E+07

0.00E+00


0 0.00001 0.00002


0.00003 0.00004 0.00005


Equivalent Mass (g)


Figure 4: (left) Individual calibration tube. (right) Plot of total photon flux vs the amount of polymer in the calibration tubes





7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010


Figure 5: Compilation picture showing a regional section of the lung model.

Table 4: Summary of regional deposition


GO 4.53% 3.77% 4.41% 4.24% + 0.41

First Carina 9.96% 9.60% 8.25% 9.27% + 0.90
G1 2.27% 3.44% 2.53% 2.75% 0.62

Second Carina 2.97% 5.46% 4.23% 4.22% + 1.25
G2 0.31% 1.55% 1.04% 0.97% + 0.62

Third Carina 13.33% 13.75% 16.21% 14.43% + 1.55

G3 & 66.64% 62.42% 63.33% 64.13% + 2.22
Connector Unit











The velocities, Stokes, Reynolds, and particle Reynolds
numbers are calculated for each airway generation, they are
listed in Table 5. The Stokes and particle Reynolds
number are based on a particle of size 2.5gm and a flow rate
of 30LPM. Looking at each region separately, there is a
significant amount of particles collecting at the trachea
region (GO). Even though the image in Figure 5 does not
show any signal in the GO region, there are still photons
being emitted. The deposition is spread over the large
surface area of the branch, which dilutes the signal. This is
somewhat unexpected. If the particles flow vertically
downward from the generation point in laminar flow, then
there should be very little amount of particles deposited at
GO. When looking at the Reynolds number for GO, the
flow is between the laminar and transition regimes. This
may account for the deposition in this area.


Table 5: Velocity, Stokes,
branch of the lung model


Velocity
[m/s)


and Reynolds number in each


Re Reparticle


0.00167 2340 0.26


2.14 0.00268 1726 0.28

2.31 0.00426 1269 0.31


2.53 0.00695 940


0.34


After the trachea, particles encounter the first
generation region, consisting of the first carina and G1
branching. The first carina area is shown to have a high
amount of deposition. This is expected the first carina is
the first obstruction point in the lung model the majority
of the particles will be filtered out at this point. There is
some deposition occurring at Gl. This can be accounted for
by the branching effect of the carina. The next section of
the lung is the second generation region, second carina and
the G2 branching. As expected, there are particles
deposited at the second carina this; these values are about
half of the particles collected at the first carina. There is
very little deposition at the G2 branching, about 1%. More
particles are expected to be deposited at the second
generation of airway, especially at the G2 branching. This
is likely due to the combined effect of the shorter length of
the branches with the particles moving at high velocity.
This reduces the residence time in the branch.

The third carina, however, has as much deposition as
the first and second carina combined. This is likely to be
caused by the small branch and the high velocity of the
particles flowing through it. The particles that were
expected to deposit at G2 may have deposited closer to this
region, causing the amount deposited at the third carina to
be higher and at G2 to be lower.

The last region in the model is the G3 & connector unit;
these two regions are consolidated into one single region.
The connector units are used to hook the model up to tubing
for the vacuum to flow through at each branch. There is an
abrupt change in diameter between the G3 branching and


7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010



the connector unit. This sudden change in diameter creates
a gap where particle are collected. This is observed by a
band of signal between the two regions. This band creates
an issue to determine the location for a fair separation
between the two regions. The two regions were grouped
together; the additional particles deposited at the gap area
would cause a skewed comparison of the G3 branching to
the other regions. This final region contains over half of
the particles deposited in the model. This number is very
high, this region contains more branches with better
separation efficiency that the previous regions. G3
branches have a very small diameter and the connector units
have even smaller diameters. The small airway sizes cause
higher deposition.

Comparison of the current data to related published
studies is rather difficult. Most published results present
the deposition efficiency of the overall model with no data
for the individual sections. A close comparison with
similar particle size and air flow rate is from a work by
Ferron (1977). In this study, deposition data were reported
for a symmetric glass model, with a mouth-throat airway
and an upper airway. The particles used were polydisperse
with mean diameter of 1.69gm and a geometric standard
deviation of 1.89. The flow rate used by Ferron was
16LPM, about half of the current study. The data
presented in the Ferron study were a plot of deposition
fraction versus the length in the lung. When integrating
the plot for the trachea, bifurcation 1, and bifurcation 2, the
resulting deposition fractions are 10%, 1.93% and 0.72%
respectively. For comparison, the deposition data needs to
be examined in a relative fashion. The ratios of the
deposition in each region to the deposition in generation 1
are 5.1, 1, and 0.375, respectively. In the current study, the
ratios are 0.35, 1, and 0.42, respectively. In the current
study, the deposition ratio of the trachea to generation 1 is
much lower; this is mainly due to the lack of the
oropharyngeal region in the upper airway. The joint
between the larynx and the trachea exhibits a sharp bend
causing a large amount of particles to deposit at the inlet of
the trachea. For the deposition ratio of generation 2 to
generation 1, the current study shows a slightly higher ratio.
This difference is explained by the higher flow rate used in
the current study and the inclusion of the smaller size
particles; larger particles would have been filtered out by
the upper airway. With lower flow rate, smaller particles
are more likely to pass the airway generation without
depositing.

There are a few concerns with the current technique,
mainly with charging issues of the particles. The particles
in this study are generated by electrospray. In this process,
particles are subject to high electrical forces. Particles are
generated with charge for this study. Because of this
charge, environment factors such as humidity have a very
large impact on the particle flow and particle-particle
interactions. The humidity had been found to cause
particles to agglomerate and to deform during generation.
Coronas are used to neutralize the particles as seen in the
schematic of the set up. The current charge measurement
mechanism can only infer whether the average charge is









positive, negative or neutral. Currently, it is not possible to
measure the exact charge of the particle. The effect of
particle charge and humidity may account for any variations
that are seen in the deposition patterns.

Setup issues aside, NIR spectroscopy is able to image
the deposition with good reproducibility. Based on the
calibration curve created by the pictures, the slope changes
very little both between pictures and day-to-day. The
background effects do change from day-to-day, but the
overall signal also changes with the background; this does
not pose as issue that hinders the use of the NIR spectrum.
Focusing on the images, pictures from the IVIS system can
be taken with good resolution. Each deposition region can
be seen clearly with a signal gradient that corresponds to a
particle concentration. Since the lung model is a 3
dimensional model, pictures were taken at different angles,
this shows the feasibility of the NIR spectrum for use with
other types of models and structures with more complex
geometry.

Conclusion

This study has shown that it is possible to use NIR
spectrum to image the regional particle deposition. The
images of different deposition trials, taken with the IVIS
system, show a good resolution and reproducibility. The
images show a brightly lit signal which can be related to a
specific section of the lung model. In addition, the
relationship of the polymer mass and photon flux changes
relatively little day-to-day and between pictures. The
results indicate that the NIR spectrum offers a safe and
non-destructive alternative approach to visualize particle
deposition in the lung and other complex morphologies.


7th International Conference on Multiphase Flow
ICMF 2010, Tampa, FL USA, May 30-June 4, 2010

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Bernhard T., Sommerauer B., Weber N., & Griese M.
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Chalupa D. C., Morrow P E., Oberdorster G, Utel M. J., &
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Dolovich M. & Labiris R. Imaging drug delivery and drug
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Eberl S., Chan H. K., Daviska E., Constable C., & Young I.
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1365-1372 (2001)

Ferron G. A. Deposition of polydisperse aerosol in two
glass model representing the upper human airway. J Aerosol
Sci. Vol. 8, 409-427 (1977)

Kreyling W. G, Demmler M., & Moller W. Dosimetry and
toxicology of ultrafine particles. J. Aerosol Med. Vol. 17,
140-152 (21 114)

Mark H. & Campbell B. An introduction to near infrared
spectroscopy and associated chemometrics. The Near
Infrared Research Corporation, (2008)

Tawhai M. H., Pullan A. J. & Hunter P. J. Generation of
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(2000)

Weibel E. R. Morphometry of the human lung. Academic
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