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This PDF file contains the front matter associated with SPIE Proceedings Volume 7904, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.
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Although femtosecond laser cell surgery is widely used for fundamental research in cell biology, the mechanisms in
the so-called low-density plasma regime are largely unknown. To date, it is still unclear on which time scales free
electron and free radical-induced chemical effects take place leading to intracellular ablation. In this paper, we
present our experimental study on the influence of laser parameters and staining on the ablation threshold. We
found that the ablation effect resulted from the accumulation of single-shot multiphoton-induced photochemical
effects finished within a few nanoseconds. In addition, fluorescence staining of subcellular structures significantly
decreased the ablation threshold. Based on our findings, we propose that dye molecules are the major source for
providing seed electrons for the ionization cascade.
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Laser microbeam has enabled highly precise non-contact delivery of exogenous materials into targeted cells, which has
been a highly challenging task while using traditional methods without compromising cell viability. We report distinct
spatial localization of impermeable substances into mammalian cells and goldfish retinal cells in explants subsequent to
ultrafast laser microbeam assisted injection, realized by focusing a near infrared tunable Ti: sapphire laser beam.
Introduction of impermeable dye into the cell through localized pore formation was confirmed by distinct fluorescence at
the site of pore formation on the membrane and its spatiotemporal diffusion pattern through the nucleus. Indirect
optoporation by bubble formation, external to cell, led to a similar spatial diffusion pattern but with a larger time
constant for injection. Using optimized laser intensity, exposure and spatial irradiation pattern, desired spatial
transfection patterns in goldfish retina explants were achieved as confirmed by expression of injected plasmids encoded
for light-activable channelrhodopsin-2 (ChR2) ion channel tagged with fluorescent protein. Laser assisted delivery of
exogenous material into specific area of three-dimensional neuronal tissue, such as the retina, will help to understand the
functioning of neuronal circuitry of normal and degenerated retina.
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Endovenous Laser Ablation (EVLA) has become a popular minimally invasive alternative to stripping in the treatment of
saphenous vein reflux. Several wavelengths have been proposed; of which 810, 940 and 980- nm are the most commonly
used. However, the most appropriate wavelength is still the subject of debate. Thermal shrinkage of collagenous tissue
during EVLA plays a significant role in the early and late results of the treatment. The aim of this study is to compare the
efficacy of 980 and 1940-nm laser wavelengths in the treatment of varicose veins. In this study, 980 and 1940-nm lasers
at different power settings (8/10W for 980-nm, 2/3W for 1940-nm) were used to irradiate stripped human veins. The
most prominent contraction and narrowing in outer and inner diameter were observed with the 1940-nm at 2W,
following 980-nm at 8W, 1940-nm at 3W and finally 980-nm at 10W. The minimum carbonization was observed with
the 1940-nm at 2W. As a conclusion, 1940-nm Tm-fiber laser which has a significant effect in the management of
varicose veins due to more selective energy absorption in water and consequently in the vein is a promising method in
the management of varicose veins.
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We explore the potential of intravascular or endoscopic optical coherence tomography (OCT) to extract relevant
mechanical properties of a tissue deformed by an inflating balloon. Tubular OCT phantoms with different mechanical
properties are fabricated. The phantoms are deformed by an inflating balloon, and the deformation is monitored with
OCT. A quantitative description of the phantom deformation is obtained by segmenting the OCT images. Two strategies
to extract the mechanical properties from this quantitative data are presented: by comparing to a finite-element
simulation and by performing a mechanical analysis.
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For a systemically administered drug to act, it first needs to cross the vascular wall. This step represents a bottleneck for
drug development, especially in the brain or retina, where tight junctions between endothelial cells form physiological
barriers. Here, we demonstrate that femtosecond pulsed laser irradiation focused on the blood vessel wall induces
transient permeabilization of plasma. Nonlinear absorption of the pulsed laser enabled the noninvasive modulation of
vascular permeability with high spatial selectivity in three dimensions. By combining this method with systemic
injection, we could locally deliver molecular probes in various tissues, such as brain cortex, meninges, ear, striated
muscle, and bone. We suggest this method as a novel delivery tool for molecular probes or drugs.
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Re-orientation of adhering cell(s) with respect to other cell(s) has not been yet possible, thus limiting study of controlled
interaction between cells. Here, we report cell detachment upon irradiation with a focused near-infrared laser beam, and
reorientation of adherent cells. The cell gets detached after irradiation for few seconds, followed by vertical orientation.
The detached cell was transported along axial direction by scattering force and trapped at a higher plane inside the media
using the same laser beam by Gravito-optical trap. The trapped cell could then be repositioned by movement of the
sample stage and reoriented by rotation of the astigmatic trapping beam. The height at which the cell was stably held was
found to depend on the laser beam power. The cell could be brought back to the substrate by reducing the laser beam
power using a polarizer or blocking the laser beam. Viability of the detached and manipulated cell was found not to be
compromised as confirmed by PI fluorescence exclusion assay. The
re-oriented cell was allowed to re-attach to the
substrate at a controlled distance and orientation with respect to other cells. Further, the cell was found to retain its shape
even after multiple detachments and manipulation using the laser beam. This technique opens up new avenues for non-contact
modification of cellular orientations that will enable study of
inter-cellular interactions and design of engineered
tissue.
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Terahertz (1012 Hz) pulsed imaging is a totally non-destructive and non-ionising imaging modality and thus potential
applications in medicine are being investigated. In this paper we present results using our hand-held terahertz probe that
has been designed for in vivo use. In particular, we use the terahertz probe to perform reflection geometry in vivo
measurements of human skin. The hand-held terahertz probe gives more flexibility than a typical flat-bed imaging
system, but it also results in noisier data and requires existing processing methods to be improved. We describe the
requirements and limitations of system geometry, data acquisition rate, image resolution and penetration depth and
explain how various factors are dependent on each other. We show how some of the physical limitations can be
overcome using novel data processing methods.
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Metal meshes work as band-pass filters in the terahertz (THz) region, with their transmission spectra acutely affected
by the refractive index of the material inside and above the metal mesh openings. We used a metal mesh for
high-sensitivity observations by focusing on the "dip", that is, a sudden change in transmittance that only appeared when
the THz wave was obliquely incident onto the metal mesh. Here we report a measurement of stratum corneum to inspect
the feasibility of applying the metal mesh sensor to observations of human skin.
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Terahertz (THz) radiation sources are now being used in a host of military, defense, and medical applications.
Widespread employment of these applications has prompted concerns regarding the health effects associated with THz
radiation. In this study, we examined the gene expression profile of mammalian cells exposed to THz radiation. We
hypothesized that if THz radiation couples directly to cellular constituents, then exposed cells may express a specific
gene expression profile indicative of ensuing damage. To test this hypothesis, Jurkat cells were irradiated with a
molecular gas THz laser (2.52 THz, 636 mWcm-2, durations: 5, 10, 20, 30, 40, or 50 minutes). Viability was assessed 24
h post-exposure using MTT assays, and gene expression profiles were evaluated 4 h post-exposure using mRNA
microarrays. Comparable analyses were also performed for hyperthermic positive controls (44°C for 40 minutes). We
found that cellular temperatures increased by ~6 °C during THz exposures. We also found that cell death increased with
exposure duration, and the median lethal dose (LD50) was calculated to be ~44 minutes. The microarray data showed that
THz radiation induced the transcriptional activation of genes associated with cellular proliferation, differentiation,
transcriptional activation, chaperone protein stabilization, and apoptosis. For most genes, we found that the magnitude of
differential expression was comparable for both the THz and thermal exposure groups; however, several genes were
specifically activated by the THz exposure. These results suggest that THz radiation may elicit effects that are not
exclusively due to the temperature rise created during THz exposures (i.e. thermal effects). In future work, we plan to
verify the results of our microarray experiments using qPCR techniques.
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Finite-difference time-domain (FDTD) methods are widely used to model the propagation of electromagnetic radiation
in biological tissues. High-performance central processing units (CPUs) can execute FDTD simulations for complex
problems using 3-D geometries and heterogeneous tissue material properties. However, when FDTD simulations are
employed at terahertz (THz) frequencies excessively long processing times are required to account for finer resolution
voxels and larger computational modeling domains. In this study, we developed and tested the performance of 2-D and
3-D FDTD thermal propagation code executed on a graphics processing unit (GPU) device, which was coded using an
extension of the C language referred to as CUDA. In order to examine the speedup provided by GPUs, we compared
the performance (speed, accuracy) for simulations executed on a GPU (Tesla C2050), a high-performance CPU (Intel
Xeon 5504), and supercomputer. Simulations were conducted to model the propagation and thermal deposition of
THz radiation in biological materials for several in vitro and in vivo THz exposure scenarios. For both the 2-D and 3-D
in vitro simulations, we found that the GPU performed 100 times faster than runs executed on a CPU, and maintained
comparable accuracy to that provided by the supercomputer. For the in vivo tissue damage studies, we found that the
GPU executed simulations 87x times faster than the CPU. Interestingly, for all exposure duration tested, the CPU,
GPU, and supercomputer provided comparable predictions for tissue damage thresholds (ED50). Overall, these results
suggest that GPUs can provide performance comparable to a supercomputer and at speeds significantly faster than
those possible with a CPU. Therefore, GPUs are an affordable tool for conducting accurate and fast simulations for
computationally intensive modeling problems.
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A two-dimensional, time-dependent bioheat model is applied to evaluate changes in temperature and water content in tissues subjected to laser irradiation. Our approach takes account of liquid-to-vapor phase changes and a simple diffusive flow of water within the biotissue. An energy balance equation considers blood perfusion, metabolic heat generation, laser absorption, and water evaporation. The model also accounts for the water dependence of tissue
properties (both thermal and optical), and variations in blood perfusion rates based on local tissue injury. Our
calculations show that water diffusion would reduce the local temperature increases and hot spots in comparison to simple models that ignore the role of water in the overall thermal and mass transport. Also, the reduced suppression of
perfusion rates due to tissue heating and damage with water diffusion affect the necrotic depth. Two-dimensional results for the dynamic temperature, water content, and damage distributions will be presented for skin simulations. It is argued that reduction in temperature gradients due to water diffusion would mitigate local refractive index variations, and hence
influence the phenomenon of thermal lensing. Finally, simple quantitative evaluations of pressure increases within the
tissue due to laser absorption are presented.
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Laser radiation has many applications in biomedical field, such as wound healing, tissue
repairing, heating and ablation processes. Intravenous low power laser radiation is used clinically for skin and vascular
disorders. Laser radiation improves microcirculation and modulates the rheological properties of blood. FTIR (Fourier
Transform Infra Red Spectra) is used to see the structural changes in erythrocyte membrane.
In the present work He Ne laser (λ= 632nm, power=2mW) is used to irradiate human Red blood cells.
Red blood cells are separated from human whole blood using centrifugation method (time=10 min., temperature=15°C
and RPM=3000) and then exposed to HeNe laser radiation. Laser exposure time is varied from 10 min. to 40min for Red
blood cells. Absorption spectrum, FTIR and fluorescence spectra of RBC are compared before and after HeNe laser
irradiation. The absorption spectrum of RBC after exposure to HeNe laser shows a significant decrease in absorbance.
The FTIR spectrum of non irradiated RBC clearly show the peaks due to O-H (free group), C=O (amide I group), N=O
(nitro group), C-O (anhydride group) and C-H (aromatic group). Laser radiation changes in transmittance in FTIR
spectra related to C=O group and percentage of transmittance increases for O-H, C=C, N=O, C-O and C-H group.
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Current laser safety standards for multiple pulse lasers are based primarily on modeling and the results of single pulse
studies. Previous thermal effects studies have focused on histological and visible endpoints, with only a few studies
examining the actual temperatures achieved. The goal of this research was to probe the actual vertical temperature profile
produced by 2.01 micron laser pulses in the cornea. In this study the corneal temperature rise from multiple 2.01 micron
Tm:YAG laser pulses was investigated using ex-vivo rabbit eyes. A thermal-measurement data set for a different number
of pulses was collected and compared. An infrared thermal camera employing microbolometer detectors captured surface
temperature rises resulting from laser pulses. Single 10 ms pulses as well as two, three, and four pulse sequences were
utilized while the total energy delivered was held constant. A comparison of the data to temperatures required for
denaturing proteins and the current laser safety guidelines will be presented.
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Laser exposure duration dictates whether tissues subjected to short visible wavelengths ( ≤ 514 nm)
are damaged by thermal (e.g. 0.1 s) or non-thermal ( ≥ 100 s) mechanisms. Somewhere between
these extremes, an abrupt transition between the two damage mechanisms has been found for both
in vitro and animal retinal models (J. Biomed. Opt. 15, 030512, 2010). Non-thermal
(photochemical) damage is characterized by an inverse relationship between damage threshold
irradiance and exposure duration (irradiance reciprocity). We have found that exposures of 40 - 60
s in an in vitro retinal model require radiant exposures well above the expected requirement for nonthermal
damage, introducing the concept that damage was forced to be thermal in mechanism.
Here we quantify and compare photo-oxidative processes at ambient temperatures between 35 - 50 °C.
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Familial Adenomatous Polyposis (FAP) is an autosomal dominant disease characterized by the development of
multiple colonic polyps at younger age with a near 100% lifetime risk of colorectal cancer in later years. The
determination of FAP is made after extensive clinical evaluation and genetic testing of at risk individuals. Genetic
testing is expensive and in some cases deleterious mutations are not found in all patients with a clinical diagnosis of
FAP. As such, the early identification of affected individuals could substantially eliminate associated morbidity and
mortality. We investigated a novel spectro-polarimetric imaging system to capture images of the oral mucosa at
different wavelengths in an attempt to distinguish patients with FAP from controls. Total diffused oral mucosal
reflectance (OMR) and oral mucosal vascular density (OMVD) were calculated from spectral data collected from 33
patients with gene positive FAP, 5 patients who tested negative for FAP, and 45 controls. A statistically significant
difference in OMVD (p < 0.001) was observed between individuals with FAP and controls. Analysis of OMR showed
no significant difference between the two subject groups.
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Light propagation in fibrous biological tissue is quite different from that in isotropic medium. Several studies
showed that the measured reduced scattering coefficient in fibrous tissue strongly depends on the measurement
direction. In this study, we investigated the possibility of retrieving optical properties in anisotropic tissue using
time-domain measurements. A Monte Carlo model was used to simulate light propagation in a fibrous tissue
consisting of aligned cylinders and spherical shape scatters. An isotropic diffuse model was then used to determine
the optical properties from simulated time-resolved reflectance. When the measurement position was parallel to the
fiber direction, the derived reduced scattering coefficient had good agreement with the true background scattering
coefficient values with a less than 10% error. In contrast, when measuring in a direction perpendicular to the
cylinders, the derived reduced scattering coefficient was close to the summation of the reduced scattering
coefficients of both cylinders and background only in samples with small cylinder diameters. If the fiber size in the
medium is known, the reduced scattering coefficient associated with the cylinders can be derived by using a
correction coefficient.
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Goals: Improving cancer diagnosis is one of the important challenges at this time. The precise differentiation
between benign and malignant tissue is in the oncology and oncologic surgery of the utmost significance. A new
diagnostic system, that facilitates the decision which tissue has to be removed, would be appreciated. In previous
studies many attempts were made to use tissue fluorescence for cancer recognition. However, no clear correlation
was found between tissue type and fluorescence parameters like time and wavelength dependent fluorescence
intensity I(t, λ). The present study is focused on cooperative behaviour of cells in benign or malignant prostates
tissue reflecting differences in their metabolism.
Material and Methods: 50 prostate specimens were obtained directly after radical prostatectomy and from
each specimen 6 punch biopsies were taken. Time-resolved fluorescence spectra were recorded for 4 different
measurement points for each biopsy. The pathologist evaluated each measurement point separately. An algorithm
was developed to determine a relevant parameter of the time dependent fluorescence data (fractal dimension DF ).
The results of the finding and the DF -value were correlated for each point and then analysed with statistical
methods.
Results: A total of 1200 measurements points were analysed. The optimal algorithm and conditions for
discrimination between malignant and non-malignant tissue areas were found. The correct classification could
be stated in 93.4% of analysed points. The ROC-curve (AUC = 0.94) confirms the chosen statistical method as
well as it informs about the specificity (0.94) and sensitivity (0.90).
Conclusion: The new method seems to offer a very helpful diagnostic tool for pathologists as well as for
surgery.
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Determination of tissue optical parameters is fundamental for application of light in either diagnostics or therapeutical
procedures. However, in samples of biological tissue in vitro, the optical properties are modified by
cellular death or cellular agglomeration that can not be avoided. This phenomena change the propagation of
light within the biological sample. Optical properties of human blood tissue were investigated in vitro at 633 nm
using an optical setup that includes a double integrating sphere system. We measure the diffuse transmittance
and diffuse reflectance of the blood sample and compare these physical properties with those obtained by Monte
Carlo Multi-Layered (MCML). The extraction of the optical parameters: absorption coefficient μa, scattering
coefficient μs and anisotropic factor g from the measurements were carried out using a Genetic Algorithm, in
which the search procedure is based in the evolution of a population due to selection of the best individual,
evaluated by a function that compares the diffuse transmittance and diffuse reflectance of those individuals with
the experimental ones. The algorithm converges rapidly to the best individual, extracting the optical parameters
of the sample. We compare our results with those obtained by using other retrieve procedures. We found that
the scattering coefficient and the anisotropic factor change dramatically due to the formation of clusters.
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Low level laser therapy (LLLT) is an emerging therapeutic approach for several clinical conditions. The clinical effects
induced by LLLT presumably go from the photobiostimulation/photobioinibition at cellular level to the molecular level.
The detailed mechanism underlying this effect is still obscure. This work is dedicated to quantify some relevant aspects
of LLLT related to molecular and cellular variations. This goal was attached by exposing malignant breast cells (MCF7)
to spatially filtered light of a He-Ne laser (633 nm) with 28.8 mJ/cm2 of fluency. The cell viability was evaluated by
microscopic observation using Trypan Blue viability test. The vibrational spectra of each experimental group (micro-
FTIR technique) were used to identify the relevant biochemical alterations occurred due the process. The red light had
influence over RNA, phosphate and serine/threonine/tyrosine bands. Light effects on cell number or viability were not
detected. However, the irradiation had direct influence on metabolic activity of cells.
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We studied a pre-charring optical behavior of blood at a laser catheter-tip during a red laser irradiation (663 nm, CW)
with around 50 W/cm2 in blood to prevent charring at the laser catheter-tip. The laser irradiated red-blood-cell shape
changes were microscopically observed. A round formation, aggregation, and hemolysis were found until blood charring
(ex vivo). A time-history of diffuse-reflected light power and transmitted light power from a thin blood layer which was
irradiated by the red laser were measured with microscope optics to investigate the charring process. The diffusereflected
light power decreased following a gentle peak before the charring. This decrease indicated the pre-charring
behavior which might be induced by scattering and absorption changes due to red-blood-cell degenerations described
above. Using the laser catheter located in porcine heart, we successfully detected the pre-charring behavior by a
backscattering light power (in vivo). We demonstrated charring prevention availability with the laser power control (ex
vivo). We think that the backscattering light power measurement and laser power control via the laser catheter might be
useful to detect pre-charring behavior, and to prevent the charring for therapeutic laser irradiation in blood under
catheterization such as arrhythmia treatment with photodynamic therapy.
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Angular Domain Imaging (ADI) has been previously demonstrated to generate projection images of attenuating targets
embedded within a turbid medium. The imaging system employs a silicon micro-tunnel array positioned between the
sample and the detection system to reject scattered photons that have deviated from the initial propagation direction and
to select for ballistic and quasi-ballistic photons that have retained their forward trajectory. Two dimensional
tomographic images can be reconstructed from ADI projections collected at a multitude of angles. The objective of this
work was to extend the system to three dimensions by collecting several tomographic images and stacking the
reconstructed slices to generate a three dimensional volume representative of the imaging target. A diode laser (808nm,
CW) with a beam expander was used to illuminate the sample cuvette. An Angular Filter Array (AFA) of 80 μm × 80
μm square-shaped tunnels 2 cm in length was used to select for image forming quasi-ballistic photons. Images were
detected with a linear CCD. Our approach was to use a SCARA robot to rotate and translate the sample to collect
sufficient projections to reconstruct a three dimensional volume. A custom designed 3D target consisting of 4 truncated
cones was imaged and reconstructed with filtered backprojection and iterative methods. A 0.5 mm graphite rod was used
to collect the forward model, while a truncated pseudoinverse was used to approximate the backward model for the
iterative algorithm.
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Conventional fluorescence imaging often does not have a mechanism to remove the scattering effect in biological tissue.
We use Angular Domain Imaging (ADI) to improve the detection of smaller structures in fluorescence layer over that
can be provided by existing systems. ADI is a high resolution, ballistic imaging method that utilizes the angular
spectrum of photons to filter multiple-scattered photons and accepts only photons with small angular deviation from their
original trajectory. Advantages of the ADI technique are that it is insensitive to wavelength and the sources are not
required to be high quality, coherent, or pulse, as with OCT or time domain. Our target is to perform fluorescence ADI at
shallow tissue such as skin (≈ 1mm) with a buried collagen layer. To experimentally model shallow tissue with phantoms,
a thin layer of scattering medium with similar scattering characteristic (μs = 200cm-1, g = 0.85) is placed on top
fluorescence plastic (415nm excitation, ≈ 555-585nm emission) which is patterned by strips of non-emitting structures
(200-400μm). Positioning multiple collimated arrays with acceptance angles of 5.71° on top of the scattering medium,
test structures (200μm wide) can be detected at shallow scattering medium thickness (1mm). Monte Carlo simulation
confirms that fluorescence ADI can image structures at shallow tissue depth by using collimator array with modest
filtration angles. Results show micromachined collimator arrays provide both high spatial resolution and angular
filtration on scattered photons.
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Light-tissue interaction is common in clinical treatments and medical researches, therefore investigation of light path in
irradiated tissues is of high importance. In this research, simulations and experimental measurements of the reflected
light intensity from one-layered lattices and phantoms are presented. Our results suggest that random walk simulations
fit well the photon migration model and enable the extraction of the lattice absorption parameter. The experimental
results present a partial fitting to the random walk model: while phantoms presenting different absorption coefficients
are distinguished by different reemitted light profiles, the model does not apply an adequate description for the phantom
absorption coefficient extraction. This calls for further investigation.
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Photothermal therapy offers a solution for the destruction of cancer cells without significant collateral damage to
otherwise healthy cells. Several attempts are underway in using carbon nanoparticles (CNPs) and nanotubes due to their
excellent absorption properties in the near-infrared spectrum of biological window. However, minimizing the required
number of injected nanoparticles, to ensure minimal cytotoxicity, is a major challenge. We report on the introduction of
magnetic carbon nanoparticles (MCNPs) onto cancer cells, localizing them in a desired region by applying an external
magnetic field and irradiating them with a near-infrared laser beam. The MCNPs were prepared in Benzene, using an
electric plasma discharge, generated in the cavitation field of an ultrasonic horn. The CNPs were made ferromagnetic by
use of Fe-electrodes to dope the CNPs, as confirmed by magnetometry. Transmission electron microscopy measurements
showed the size distribution of these MCNPs to be in the range of 5-10 nm. For photothermal irradiation, a tunable
continuous wave Ti: Sapphire laser beam was weakly focused on to the cell monolayer under an inverted fluorescence
microscope. The response of different cell types to photothermal irradiation was investigated. Cell death in the presence
of both MCNPs and laser beam was confirmed by morphological changes and propidium iodide fluorescence inclusion
assay. The results of our study suggest that MCNP based photothermal therapy is a promising approach to remotely
guide photothermal therapy.
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In the present work, we demonstrate a potential use of gold nanorods as a contrast agent for selective photothermal
therapy of human acute leukemia cells (HL-60) using a near-infrared laser. Gold Nanorods (GNR) are synthesized
and conjugated to CD33, a 67 kDa glycoprotein found on the surface of myeloid cells that belongs to the
sialoadhesin family of proteins. After pegylation, or conjugation with CD33 antibody, GNR were non-toxic for
acute and chronic leukemia cells. We used a Quanta System q-switched titanium sapphire laser emitting at a center
wavelength of 755 nm. Each sample was illuminated with 1 laser shot at either high or low fluence. Both laser
modes were used in 3 independent cell probes. HL-60 cells were treated for 45 min with GNR conjugated with mAb
CD33, or with GNR-Pegylated particles. After laser application, the cells were resuspended and analyzed to cell
viability with Trypan blue exclusion assay. GNR-CD33 conjugates significantly increase the percentage of cell
death as compared with a control group after laser illumination: a 3 fold increase is observed.
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ABSTRACT
Damage thresholds (ED50) for skin using Yucatan mini-pig (Sus scrofa domestica)
have been determined at the operational wavelength of 1319 nm with beam diameters of
0.61 cm and 0.96 cm. Exposure durations of 0.25, 1.0, 2.5 and 10 seconds were used to
determine trends in damage threshold with respect to exposure time and beam diameter at
this moderately-high penetrating wavelength. A relatively narrow range of total radiant
exposure from 37.4 J/cm2 to 62.3 J/cm2 average was observed for threshold damage with
laser parameters encompassing a factor of two in beam area and a factor of forty in
exposure duration.
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We report on an investigation aimed to increase the efficiency of photodynamic therapy (PDT) through the influence of
localized surface plasmon resonances (LSPR's) in metal nanoparticles. PDT is based on photosensitizers that generate
singlet oxygen at the tumour site upon exposure to visible light. Although PDT is a well-established treatment for skin
cancer, a major drawback is the low quantum yield for singlet-oxygen production. This motivates the development of
novel methods that enhance singlet oxygen generation during treatment. In this context, we study the photodynamic
effect on cultured human skin cells in the presence or absence of gold nanoparticles with well established LSPR and
field-enhancement properties. The cultured skin cells were exposed to protoporphyrin IX and gold nanoparticles and
subsequently illuminated with red light. We investigated the differences in cell viability by tuning different parameters,
such as incubation time and light dose. In order to find optimal parameters for specific targeting of tumour cells, we
compared normal human epidermal keratinocytes with a human squamous skin cancer cell line. The study indicates
significantly enhanced cell death in the presence of nanoparticles and important differences in treatment efficiency
between normal and tumour cells. These results are thus promising and clearly motivate further development of
nanoparticle enhanced clinical PDT treatment.
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Balb/c wild type mice were used to perform in vivo experiments of laser-induced thermal damage to the retina. A
Heidelberg Spectralis HRA confocal scanning laser ophthalmoscope with a spectral domain optical coherence
tomographer was used to obtain fundus and cross-sectional images of laser induced injury in the retina. Sub-threshold,
threshold, and supra-threshold lesions were observed using optical coherence tomography (OCT), infrared reflectance,
red-free reflectance, fluorescence angiography, and autofluorescence imaging modalities at different time points post-exposure.
Lesions observed using all imaging modalities, except autofluorescence, were not visible immediately after
exposure but did resolve within an hour and grew in size over a 24 hour period. There was a decrease in fundus
autofluorescence at exposure sites immediately following exposure that developed into hyper-fluorescence 24-48 hours
later. OCT images revealed threshold damage that was localized to the RPE but extended into the neural retina over a 24
hour period. Volumetric representations of the mouse retina were created to visualize the extent of damage within the
retina over a 24 hour period. Multimodal imaging provides complementary information regarding damage mechanisms
that may be used to quantify the extent of the damage as well as the effectiveness of treatments without need for
histology.
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Even though catheterization or electric stimulation are used for treatment of neurogenic bladder, invasiveness and
inconvenience of these approaches prompt us to develop a new possible therapeutic method to control urination by using
optical stimulation. The optical method using femtosecond pulsed laser (FSPL) has advantages of focused and
subsurface stimulation. Irradiation of FSPL induced a rapid increase of intracellular calcium level followed by
contraction of primary cultured human bladder smooth muscle cells. Short exposure of bladder detrusor ex-vivo to FSPL
also induced a controlled contraction of detrusor. Collectively, we propose that FSPL can be considered as a potential
therapeutic approach for intractable neurogenic bladder.
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We were unable to reproduce published inactivation results, or show any interaction, between 90 femtosecond (fs) pulses
of 850 nm or 425 nm laser radiation and buffer/water, DNA, protein, M13 bacteriophage or E. coli. Using agarose
electrophoresis and polyacrylamide gel electrophoresis, we examined purified plasmid DNA (pUC19), bovine serum
albumin, and DNA and coat proteins extracted from M13 following exposures to irradiances of up to 120 MW/cm2. We
measured M13 viability using an assay for plaque-forming ability in soft agar after exposure to the same irradiances used
for the protein and DNA experiments. Exposures of up 1 GW/cm2 at 850 nm had no effect on the viability of E. coli as
measured by a colony forming assay in soft agar. Peroxynitrite, known to be toxic, to cause single strand breaks in
DNA, and fragment proteins in vitro gave positive results in all assays.
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Computational molecular docking simulations (Dock and AutoDock) may provide a
wealth of structural information related to the bound configuration of protein-ligand
complexes, but they require verification to ensure their results correctly predict the
bound complex. Resonance Raman spectroscopy data has been collected to correlate
normal mode vibrations observed in the bound configurations to computationally generated
structures in order to determine the best match between the in silico model
and experiment. This methodology was used to determine the bound structures at
an atomistic level of β-lactoglobulin (BLG) and meso-tetrakis (p-sulfonatophenyl) porphyrin
(TSPP) in aqueous solutions at pH 7 and 9. Comparisons of Raman spectra of
TSPP before and after binding to BLG yield line shifts that are generated by the noncovalent
binding of the ligand to the protein. Previous studies have shown that the
Tanford transition in BLG, which occurs above pH 7.9, destabilizes the protein, allowing
it to undergo a laser-induced structural change when bound to TSPP and illuminated by
at least 0.3 J of laser energy. By examining the structures at pHs above and below the
transition, we hope to reveal the mechanism of action that initiates the laser-induced
changes in the protein. Future studies will use the computed bound configuration as
an initial condition for molecular dynamics simulations of the laser-protein-complex
interaction to predict the final state of the protein after irradiation.
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Fluorescent imaging of cells and tissues cultured within a rotating wall vessel bioreactor offers quantitative assessment
of the 3-dimensional aggregation of cells into tissue constructs. We present the design of a rotating wall vessel system
optimized for real-time fluorescent analysis. The modulation transfer function of our system is found to be superior to
the commercially-available vessel used in previous fluorescence imaging studies. We demonstrate dynamic fluorescent
imaging of DAPI-stained porcine pancreatic islets.
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Tissue scaffolds are an integral part of the tissue engineering process, assisting in the culturing of cells in three
dimensions. It is important to understand both the properties of the scaffold and the growth of cells within the scaffold.
This paper describes a system to characterise scaffolds using acoustic techniques alone and the development of an
ultrasound modulated optical tomography system to study the growth of cells within the scaffolds.
Our interest is in characterising the properties of gel-based and polymer foam-based scaffolds. Results from a purely
acoustic system have been used to investigate the properties of foam scaffolds manufactured from synthetic polyesters
poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) via a supercritical fluid process. As these are porous
materials, they are particularly challenging acoustically as the pores scatter sound significantly. However, it is
demonstrated that acoustic signals are detectable through a 6mm thick scaffold.
Although acoustics alone can be used to characterize many properties of the scaffolds, useful information can also be
obtained from optical techniques e.g. monitoring the growth of cells within the scaffold via optical absorption or
fluorescence techniques. Light scattering is of course a significant problem for relatively thick engineered tissue
(~5mm). The acoustic approach has been extended to include laser illumination and detection of the ultrasound
modulated optical pulse. Images of optically-absorbing materials embedded in gel-based tissue phantoms will be
presented demonstrating that a lateral resolution of 250μm and an axial resolution of ~90μm can be achieved in
scattering samples.
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With the increasing complexity of the chemical composition of pharmaceuticals, cosmetics and everyday substances, the
awareness of potential health issues and long term damages for humanoid organs is shifting into focus. Artificial in vitro
testing systems play an important role in providing reliable test conditions and replacing precarious animal testing.
Especially artificial skin equivalents ASEs are used for a broad spectrum of studies like penetration, irritation and
corrosion of substances. One major challenge in tissue engineering is the qualification of each individual ASE as in vitro
testing system. Due to biological fluctuations, the stratum corneum hornified layer of some ASEs may not fully develop
or other defects might occur. For monitoring these effects we developed an fully automated Optical Coherence
Tomography device. Here, we present different methods to characterize and evaluate the quality of the ASEs based on
image and data processing of OCT B-scans. By analysing the surface structure, defects, like cuts or tears, are detectable.
A further indicator for the quality of the ASE is the morphology of the tissue. This allows to determine if the skin model
has reached the final growth state. We found, that OCT is a well suited technology for automatically characterizing
artificial skin equivalents and validating the application as testing system.
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Aligned, electrospun fibers have been used in a wide variety of applications from filters to scaffolds for tissue
engineering. In this study we demonstrate a quick and accurate method to quantify fiber alignment using the Radon
Transform. To test the accuracy of this method, we generated mock images fibers with varying degrees of fiber
alignment. Images were filtered to detect edges and analyzed with the Radon Transform from 1 to 180 degrees at 1
degree intervals. The absolute values of each column were summed and used to create a normalized probability
distribution function. The probability distribution function was quantified using both the full width half- maximum
(FWHM) and calculating the entropy of the function. These results were compared to an analysis method using the fast
Fourier transform. The FWHM for the Radon transform was consistent and statistically different at all fiber orientations
for different degrees of fiber variation. Both the entropy analysis for the Radon transform and the FWHM for the fast
Fourier transform did not show statistical difference. The FWHM method for the radon transform was performed on
electrospun fibers and showed statistical difference between two groups known to be statistically different by manual
analysis.
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In 1999 we have introduced a new approach for treatment of spine diseases based on the mechanical effect of nondestructive
laser radiation on the nucleus pulposus of the intervertebral disc. Laser reconstruction of spine discs (LRD)
involves puncture of the disc and non-destructive laser irradiation of the nucleus pulposus to activate reparative
processes in the disc tissues. In vivo animal study has shown that LRD allows activate the growth of hyaline type
cartilage in laser affected zone. The paper considers physical processes and mechanisms of laser regeneration, presents
results of investigations aimed to optimize laser settings and to develop feedback control system for laser reparation in
cartilages of spine and joints. The results of laser reconstruction of intervertebral discs for 510 patients have shown
substantial relief of back pain for 90% of patients. Laser technology has been experimentally tested for reparation of
traumatic and degenerative diseases in joint cartilage of 20 minipigs. It is shown that laser regeneration of cartilage
allows feeling large (more than 5 mm) defects which usually never repair on one's own. Optical techniques have been
used to promote safety and efficacy of the laser procedures.
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Red blood cells (RBC) possess unique viscoelastic characteristics which allow them to pass through capillaries
narrower than their size. Measurement of viscoelastic property of cells (e.g. RBC) in low-force regime is of high
significance as it represents conditions of membrane fluctuation in response to physiological conditions.
Estimation of visco-elastic properties of RBC requires measurement of extent of deformation in RBC subjected
to known force. Optical tweezers, being gentle and absolutely sterile, are emerging as the tool of choice for
application of localized force on cells. However, stretching of RBC in very low force regime has not been
quantified. Further, though deformations in transverse directions have been measured, vertical deformations due
to stretching of cells cannot be quantified by classical microscopic images. Here, we report realization of offaxis
digital holographic microscopy (DHM) for highly sensitive axial changes in RBC shape due to stretching
by optical tweezers without attaching microscopic beads. The RBC was stretched in axial direction with
nanometer precision by change of divergence of the trapping beam. The obtained deformation patterns were
compared with the axial position of the tweezers focus. Since the pathophysiology of progression of diseases
like malaria and cancer is reflected in the biophysical (both mechanical and material) properties of the cells, it is
possible to identify the changes by simultaneous measurement of refractive index and elasticity using this
approach.
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We firstly investigate the mechanic and acoustic properties of human teeth by using the laser
generation of surface acoustic wave (SAW) technique. The materials investigated included
normal and decayed teeth, which have the same grain size and different thickness, are used as
the samples. The tissue responds to the laser-induced stress by thermoelastic expansion. We
can obtain the shape of acoustic pulse and the phase velocity was determined for the teeth
system and extract information on the teeth thickness, density, and transverse sound velocity
that could be used as diagnostic parameters.
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Vascular endothelial growth factor (VEGF) is known for its role in neovascularization and cellular signaling pathways of
sub-threshold retinal lesions. The objective of this study was to elucidate potential protection mechanisms to laser-induced
heat stress utilizing an in vitro retinal model. The cell line was characterized to determine the relative abundance
of VEGF-C protein. Cells, preconditioned via water bath and controls, were then exposed to 2 μm laser radiation to
assess whether increases in protein production following preconditioning could confer any protection. There was no
significant increase in threshold damage irradiance (ED50) in the preconditioned cells versus control.
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Uneven distribution of skin color is one of the biggest concerns about facial skin appearance. Recently several
techniques to analyze skin color have been introduced by separating skin color information into chromophore
components, such as melanin and hemoglobin. However, there are not many reports on quantitative analysis of
unevenness of skin color by considering type of chromophore, clusters of different sizes and concentration of the each
chromophore. We propose a new image analysis and simulation method based on chromophore analysis and spatial
frequency analysis. This method is mainly composed of three techniques: independent component analysis (ICA) to
extract hemoglobin and melanin chromophores from a single skin color image, an image pyramid technique which
decomposes each chromophore into multi-resolution images, which can be used for identifying different sizes of
clusters or spatial frequencies, and analysis of the histogram obtained from each multi-resolution image to extract
unevenness parameters. As the application of the method, we also introduce an image processing technique to change
unevenness of melanin component. As the result, the method showed high capabilities to analyze unevenness of each
skin chromophore: 1) Vague unevenness on skin could be discriminated from noticeable pigmentation such as freckles
or acne. 2) By analyzing the unevenness parameters obtained from each multi-resolution image for Japanese ladies, agerelated
changes were observed in the parameters of middle spatial frequency. 3) An image processing system
modulating the parameters was proposed to change unevenness of skin images along the axis of the obtained age-related
change in real time.
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Monte Carlo (MC) simulation was implemented in a three dimensional tooth model to simulate the light propagation in
the tooth for antibiotic photodynamic therapy and other laser therapy. The goal of this research is to estimate the light
energy deposition in the target region of tooth with given light source information, tooth optical properties and tooth
structure. Two use cases were presented to demonstrate the practical application of this model. One case was comparing
the isotropic point source and narrow beam dosage distribution and the other case was comparing different incident
points for the same light source. This model will help the doctor for PDT design in the tooth.
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Angular Domain Spectroscopic Imaging employs an array of micro-channels to perform angular filtering of light that
traverses a turbid sample to reject moderately to highly scattered light. In this work, we experimentally characterized an
ADSI system by measuring transmission spectra and the first and second derivatives obtained from absorbing and
scattering targets. The derivative analysis was used to estimate the concentration of indocyanine green mixed in a
scattering liquid. The experimental results provided support for ADSI as a potential method for quantitative
spectroscopic imaging of ex vivo tissue samples.
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In this research, we present the first multi-GPU FDTD implementation of Maxwell's equations in dispersive media that
uses the Open-MP API to synchronize the operation of GPUs and their corresponding CPUs. By taking advantage of the
CUDA programming model, we present a unique implementation of the FDTD scheme that exploits the memory
hierarchy of GPUs, including the global, texture, and shared memory. This enables us to tackle problems that are
otherwise computationally prohibitive. Practical results will be presented along with a measure of speedup factors
achieved when using multiple GPU processors.
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The aim of this work was to establish measurement conditions under which endogenous skin fluorescence ("auto-fluorescence")
is relatively invariant, so that changes in exogenous agents can be accurately determined. Fluorescence
emission was measured on the volar forearm of 36 subjects, chosen to be equally representative of all 6 Fitzpatrick skin
types. All subjects were exposed to approximately 40 minutes of optical excitation at 450 and 500 nm with 4 irradiances
between 0.3 and 9 mW/cm2. Both non-optically-induced (e.g. tissue settling and fluctuation) and optically-induced
variations were observed in the measured fluorescence and mechanisms explaining these effects are proposed. The
optically-induced auto-fluorescence decay was independent of skin type when excited at 450 nm, but significantly
dependent on skin type when excited at 500 nm. Further, the extent of decay over time was linearly related to irradiance
at 500 nm, but at 450 nm was non-linear, with the extent of decay rolling off between 2 and 9 mW/cm2. In order to
maintain the auto-fluorescence signal within 95% of its original value over a 30 minute period, the excitation at 450 nm
would need to be limited to 1.5 mW/cm2, while excitation at 500 nm should be limited to 5 mW/cm2.
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The detailed mechanism of blast-induced traumatic brain injury (bTBI) has not been revealed yet. Thus, reliable
laboratory animal models for bTBI are needed to investigate the possible diagnosis and treatment for bTBI. In this study,
we used laser-induced shock wave (LISW) to induce TBI in rats and investigated the histopathological similarities to
actual bTBI. After craniotomy, the rat brain was exposed to a single shot of LISW with a diameter of 3 mm at various
laser fluences. At 24 h after LISW exposure, perfusion fixation was performed and the extracted brain was sectioned; the
sections were stained with hematoxylin-eosin. Evans blue (EB) staining was also used to evaluate disruption of the blood
brain barrier. At certain laser fluence levels, neural cell injury and hemorrhagic lesions were observed in the cortex and
subcortical region. However, injury was limited in the tissue region that interacted with the LISW. The severity of injury
increased with increasing laser fluence and hence peak pressure of the LISW. Fluorescence originating from EB was
diffusively observed in the injuries at high fluence levels. Due to the grade and spatial controllability of injuries and the
histological observations similar to those in actual bTBI, brain injuries caused by LISWs would be useful models to
study bTBI.
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To estimate the error of scattering coefficient spectrum determined by using double-integrating sphere system and
inverse Monte Carlo method, optical properties of tissue phantom were measured. The tissue phantom was composed of
hemoglobin, intralipid and gelatin. The thickness of samples
(0.1-1.0 mm) and hemoglobin concentration (0.5-4.0
mg/ml) were changed and the effects of optical properties spectra were investigated. As the results, when the value of μa
was large, μ's spectrum was not consistent with scattering theory. The higher hemoglobin concentration of samples was
the lager the errors of μ's spectra were. The thinner the sample was, the smaller the errors were. However μa spectrum
was not accurate when the sample was thin. It was predicted that when the sample thickness was 0.1 mm μ's spectrum
was accurate. And when the sample thickness was 1.0 mm, μa spectrum was accurate.
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When imaging through small aquatic creatures, scattered photons produce problems in image quality and resolution.
Angular Domain Imaging (ADI) reduces scattered photons and improves the image quality and resolution. ADI is an
imaging technique which utilizes the angular spectrum of photons to filter multiple-scattered photons and accept only
photons with small angular deviation from their original trajectory. Advantages of the ADI technique are that it is
insensitive to wavelength and the sources are not required to be high optical quality, coherent, or pulsed, as with OCT or
time domain. Our target is to image a small species called Branchiostoma lanceolatum, a lancet that is 5-8cm long and
5mm thick, by using ADI to remove the scattering in order to image internal structures. A laser illuminates the lancelet
in a water-filled container and a spatiofrequency filter removes the scattered photons before the imager. Experimentally,
a coherent Nd:Yag second harmonic (533nm) laser creates images but also optical interference occuring within the
internal structures of the lancelet. Conversely, an incoherent
broad-band white light source eliminates the structural
interference effect; however, the wavelength variation of the scattering coefficient combined with the limitation of the
image sensor's dynamic range limit the ability to distinguish the internal structures in many areas. Thus, an IR diode
laser (780nm) is used to lower the scattering coefficient as compared to conventional visible light source and to diminish
the interference effects due to its shorter coherence length.
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The function of a protein is correlated to its structure. Thus, the ability to control the structure by
unfolding the protein becomes of considerable interest. One novel method of unfolding a protein involves
using a light activated ligand bound to the protein which then triggers a photochemical reaction. Many
porphyrins are natural products and are cell-friendly within certain limits of concentration. Many of them
have been used for Photodynamic Therapy (PDT) of cancer. The specific porphyrin used in this study is
meso-tetrakis (sulfonatophenyl) porphyrin (TSPP). In this study we investigated the binding of TSPP to
Tubulin and the effects of irradiating the porphyrin/protein complex in an attempt to induce unfolding of
Tubulin. Tubulin is an important protein which forms microtubules and has been the target of many
anticancer therapies. A combination of various spectroscopic methods can be used to gain insight into the
structural changes induced by the photosensitizer on the protein and provide a blueprint of the molecular
interactions. Absorption and fluorescence spectroscopy yields information on how the electronic
transition energy levels may be changing. Resonance Raman spectroscopy (RRS) provides structural
information based on the changes of the vibrational modes of the ligand and circular dichroism (CD)
probes the secondary structure of the protein. Thus by taking spectroscopic measurements of the
protein/porphyrin complex before and after irradiation we can obtain structural information of the effects
induced by light absorption. The results indicate that there is indeed significant unfolding of Tubulin as a
result of irradiating the bound Porphyrin.
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