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- Front Matter: Volume 7561
- Fluorescence and Raman Diagnosis I
- Quantum Coherent Effects in Biology and Medicine
- Fluorescence and Raman Diagnosis II
- Imaging Diagnosis
- Biophotonic Approach for Disease and Virus Detection
- Poster Session
Front Matter: Volume 7561
Front Matter: Volume 7561
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This PDF file contains the front matter associated with SPIE Proceedings Volume 7561, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.
Fluorescence and Raman Diagnosis I
Visualization of epithelial morphology using autofluorescence microscopy under ultraviolet excitation
Show abstract
Epithelial microstructures related to early stage carcinoma are currently invisible to traditional white light endoscopy.
We recently demonstrated that autofluorescence microscopy under ultraviolet excitation can visualize superficial
microstructures without the use of contrast agents, sectioning methods, or tissue preparation. Spectroscopic analysis
allowed a better understanding of autofluorescence signal characteristics at the microscopic level and the mechanism for
achieving high quality imaging of the superficial epithelial layer with conventional wide-field microscopy. The
designing parameters for the adaptation of this technology into an endoscope probe for real-time in vivo microscopy are
tested using a bench-top prototype system. This approach may provide a powerful tool for the detection and staging of
carcinomas.
Advances in handheld spectral sensors and systems
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Spectrometer system designs have evolved rapidly over the last decade after a major paradigm shift occurred as spectroscopy
systems advanced from bulky lab based instruments to the modern compact, flexible, and portable instruments
we see today. Previously, these complicated tabletop laboratory instruments required controlled conditions to function
and were extremely expensive. That changed with the introduction of compact fiber coupled microspectrometers that
combined innovative compact designs with low-cost detectors developed for high volume commercial applications.
The miniature spectrometer dramatically broadened the applications and markets for spectroscopy. No longer did users
have to carry the sample to the spectrometer, now they could take the spectrometer to the sample enabling thousands of
new applications. Over time, the performance and benefits of these compact systems have improved. The recent development
of CMOS sensors and imagers and extremely powerful compact microprocessors has enabled a new phase of
even more compact spectroscopy systems.
UV extended supercontinuum source for time resolved and steady state spectroscopy for biological and chemical molecules
Show abstract
The spectrum of the supercontinuum generated by a femtosecond Ti:Sapphire laser beam in photonic crystal fiber (PCF)
is increased into the UV using small core diameter PCF with zero dispersion wavelength (ZDW) shorter 600 nm. A flat
spectrum is generated that spans from 350 to 1000 nm. The SC was used as an excitation source for fluorescence
spectroscopy. Fluorescence spectra can be detected from dye molecules, and native molecules in tissues samples with
excitation from wavelengths extracted from ultrafast SC light in the spectral range between 350 to 500 nm using narrow
bandpass filters. A Streak Camera was used for time-resolved fluorescence measurements.
Optical spectroscopy approach for the predictive assessment of kidney functional recovery following ischemic injury
Show abstract
Tissue that has undergone significant yet unknown amount of ischemic injury is frequently encountered in organ
transplantation and trauma clinics. With no reliable real-time method of assessing the degree of injury incurred in tissue,
surgeons generally rely on visual observation which is subjective. In this work, we investigate the use of optical
spectroscopy methods as a potentially more reliable approach. Previous work by various groups was strongly suggestive
that tissue autofluorescence from NADH obtained under UV excitation is sensitive to metabolic response changes. To
test and expand upon this concept, we monitored autofluorescence and light scattering intensities of injured vs. uninjured
rat kidneys via multimodal imaging under 355 nm, 325 nm, and 266 nm excitation as well as scattering under 500 nm
illumination. 355 nm excitation was used to probe mainly NADH, a metabolite, while 266 nm excitation was used to
probe mainly tryptophan to correct for non-metabolic signal artifacts. The ratio of autofluorescence intensities derived
under these two excitation wavelengths was calculated and its temporal profile was fit to a relaxation model. Time
constants were extracted, and longer time constants were associated with kidney dysfunction. Analysis of both the
autofluorescence and light scattering images suggests that changes in microstructure tissue morphology, blood
absorption spectral characteristics, and pH contribute to the behavior of the observed signal which may be used to obtain
tissue functional information and offer predictive capability.
Rotational dynamics and polarization anisotropy of bound and unbound receptor-targeted contrast agents in cancerous and normal prostate tissues studied by time-resolved fluorescence spectroscopy
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Temporal profiles of polarized fluorescence emitted from receptor-targeted contrast agents: Cybesin and Cytate, in
prostate tissues were studied using ultrafast time-resolved spectroscopy. An analytical model was developed and used to
investigate rotational dynamics and fluorescence polarization anisotropies of the contrast agents in prostate tissues from
the measured data. The differences of rotational times and polarization anisotropies were observed for Cybesin (Cytate)
in cancerous and normal prostate tissues, which reflect changes of micro-structures of cancerous and normal tissues, and
their different bound affinity with contrast agents. This research may be used to develop better optical methods for in situ
prostate cancer detection.
Stokes shift spectroscopy for breast cancer diagnosis
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The objective of this study is to assess the diagnostic potential of stokes shift (SS) spectroscopy (SSS) for normal and
different pathological breast tissues such as fibroadenoma and infiltrating ductal carcinoma. The SS spectra is measured
by simultaneously scanning both the excitation and emission wavelengths while keeping a fixed wavelength interval
Δλ=20 nm between them. Characteristic, highly resolved peaks and significant spectral differences between normal and
different pathological breast tissues were observed. The SS spectra of normal and different pathological breast tissues
shows the distinct peaks around 300, 350, 450, 500 and 600 nm may be attributed to tryptophan, collagen, NADH, flavin
and porphyrin respectively. Using SSS technique one can obtain all the key fluorophores in a single scan and hence they
can be targeted as a tumor markers in this study. In order to quantify the altered spectral differences between normal and
different pathological breast tissues are verified by different ratio parameters.
Quantum Coherent Effects in Biology and Medicine
From Förster resonance energy transfer to coherent resonance energy transfer and back
Robert M. Clegg,
Melih Sener,
. Govindjee
Show abstract
Photosynthesis converts solar energy into chemical energy. It provides food and oxygen; and, in the future, it
could directly provide bioenergy or renewable energy sources, such as bio-alcohol or hydrogen. To exploit such a highly
efficient capture of energy requires an understanding of the fundamental physics. The process is initiated by photon
absorption, followed by highly efficient and extremely rapid transfer and trapping of the excitation energy. We first
review early fluorescence experiments on in vivo energy transfer, which were undertaken to understand the mechanism
of such efficient energy capture. A historical synopsis is given of experiments and interpretations by others that dealt
with the question of how energy is transferred from the original location of photon absorption in the photosynthetic
antenna system into the reaction centers, where it is converted into useful chemical energy. We conclude by examining
the physical basis of some current models concerning the roles of coherent excitons and incoherent hopping in the
exceptionally efficient transfer of energy into the reaction center.
Quantum effects in biological systems
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Identification of non-trivial quantum mechanical effects in the functioning of biological systems has been a
long-standing and elusive goal in the fields of physics, chemistry and biology. Recent progress in control and
measurement technologies, especially in the optical spectroscopy domain, have made possible the identification
of such effects. In particular, electronic coherence was recently shown to survive for relatively long times in
photosynthetic light harvesting complexes despite the effects of noisy biomolecular environments. Motivated by
this experimental discovery, several recent studies have combined techniques from quantum information, quantum
dynamical theory and chemical physics to characterize the extent and nature of quantum dynamics in light
harvesting structures. I will review these results and summarize our understanding of the subtle quantum effects
in photosynthetic complexes. Then I will outline the remarkable properties of light harvesting complexes that
allow quantum effects to be significant at dynamically relevant timescales, despite the decohering biomolecular
environment. Finally, I will conclude by discussing the implications of quantum effects in light harvesting
complexes, and in biological systems in general.
Fluorescence and Raman Diagnosis II
Telegrapher-based fluorescence-enhanced optical tomography in small volume
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Accurate modeling of photon propagation in small animals is critical to quantitatively obtain accurate
tomographic images. The diffusion approximation is used for biomedical optical diagnostic techniques in turbid
large media where absorption is low compared to scattering system. This approximation has considerable limitations
to accurately predict radiative transport in turbid small media and also in a media where absorption is high compared
to scattering systems. A radiative transport equation (RTE) is best suited for photon propagation in human tissues.
However, such models are quite expensive computationally. To alleviate the problems of the high computational
cost of RTE and inadequacies of the diffusion equation in a small volume, we use telegrapher equation (TE) in the
frequency domain for fluorescence-enhanced optical tomography problems. The telegrapher equation can
accurately and efficiently predict ballistic as well as diffusion-limited transport regimes which could simultaneously
exist in small animals. The telegrapher-based model is tested by comparing with the diffusion-based model using
stimulated data in a small volume. This work shows the telegrapher-based model is appropriate in small animal
optical tomography problems.
Polarized fluorescence study in human cervical tissue: change in autofluorescence through different excitation wavelengths
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This study aims towards applying the intrinsic fluorescence technique, extracted from polarized fluorescence, to
detect subtle biochemical changes occurring during the progression of cancer from human cervical tissue samples. The
efficacy of this technique, earlier validated through tissue phantoms, is tested in human cervical tissues by comparing the
biochemical changes for diagnostic purpose at different wavelengths. It is pertinent to note that the co and crosspolarized
fluorescence do not display the high sensitivity obtained through extracted intrinsic fluorescence. We observed
that sensitivity and specificity of intrinsic fluorescence technique is high at 325 and 370nm for Collagen and NADH
respectively in comparison to 350nm excitation wavelength. It may be concluded that decoupled information at 325 and
370nm wavelengths for collagen and NADH respectively, through intrinsic fluorescence provides better diagnostic
parameter for early detection of cervical dysplasia. This information can provide a guiding path for designing a probe for
clinical purpose.
Imaging Diagnosis
Multimodality optical imaging combining optical coherence tomography (OCT) and fluorescence lifetime imaging (FLIM) for morphological and biochemical tissue characterization
Show abstract
Here we report on our current efforts to simultaneously quantify both morphological and biochemical tissue information
by combining optical coherence tomography (OCT) and fluorescence lifetime imaging (FLIM). The Fourier domain
OCT module is built around a custom designed high-speed spectrometer (bandwidth of 102 nm, 3 dB rolloff of 1.2 mm,
lines rates of up to 59 kHz). A 40 nm bandwidth SLED centered at 830 nm provided an axial resolution of 7.6 mm for
OCT. The objective lens provided 10 um lateral resolution for OCT and 100 um for FLIM. Lateral OCT and FLIM beam
scanning was accomplished using a set of galvo mirrors. The FLIM module excites and collects the fluorescence decay
signal pixel by pixel coincident with OCT A-line collection. Each 2-D FLIM image has a corresponding coregistered
OCT volume. Fluorescence excitation for FLIM was provided by a solid-state pulse laser (355 nm, 1 ns FWHM, 50 kHz
rep rate). The fluorescence signal was detected with a MCP-PMT coupled to a 1.5 GHz digitizer (250 ps temporal
resolutions). In addition, simultaneous multispectral time-resolved fluorescence detection was achieved by separating the
fluorescence emission in three bands using a series of dichroic mirrors and bandpass filters, and launching each band into
three fibers of different lengths (providing a time delay of 50 ns among bands) focused onto the MCP-PMT. The
resulting OCT/FLIM system is capable of a maximum A-line rate of 59 kHz for OCT and a maximum pixel rate of at
least 30 kHz for FLIM. The multimodality OCT/FLIM imaging system was validated on biological tissue. Future efforts
include evaluating its potential for oral cancer diagnosis and intravascular imaging.
Multispectral imaging techniques observing the dynamic changes in the hemoglobin concentrations as diagnostic tool for diseased tissues
Show abstract
Tissue oxygenation imaging is a promising diagnostics tool to study the changes and dynamics of tissue perfusion
reflecting pathologic and/or physiologic conditions of tissue. In clinical settings, imaging of local oxygenation or blood
perfusion variations can be useful for e.g. detection of skin cancer, detection of early inflammation, effectiveness of
peripheral nerve block anesthesia, study of the process of wound healing or localization of the cerebral area causing an
epileptic attack. In this study, two oxygenation imaging methods based on multi-spectral techniques were evaluated: one
system consisting of a CCD camera in combination with a Liquid Crystal Tunable Filter (420 - 730 nm or 650-1100 nm)
and a broad band (white) light source, while the second system was a CCD camera in combination with a tunable multispectral
LED light source (450-890nm).
By collecting narrowband images at selected wavelengths, concentration changes of the different chromophores at the
surface of the tissue (e.g. dO2Hb, dHHb and dtHb) can be calculated using the modified Lambert Beer equation. Two
analyzing methods were used to calculate the concentration changes this to reduce the errors caused by movement of the
tissue. In vivo measurements were obtained during skin oxygen changes induced by temporary arm clamping to validate
the methods and algorithms. Functional information from the tissue surface was collected, in non-contact mode, by
imaging the hemodynamic and oxygenation changes just below that surface. Both multi-spectral imaging techniques
show promising results for detecting dynamic changes in the hemoglobin concentrations. The algorithms need to be
optimized and image acquisition and processing needs to be developed top real time for practical clinical applications.
Tissue characterization by using narrow band imaging
Show abstract
NBI (Narrow Band Imaging) was first introduced in the market in 2005 as a technique enabling to enhance image
contrast of capillaries on a mucosal surface(1). It is classified as an Optical-Digital Method for Image-Enhanced
Endoscopy(2). To date, the application has widely spread not only to gastrointestinal fields such as esophagus, stomach
and colon but also the organs such as bronchus and bladder. The main target tissue of NBI enhancement is capillaries.
However, findings of many clinical studies conducted by endoscopy physicians have revealed that NBI observation
enables to enhance more other structures in addition to capillaries. There is a close relationship between those enhanced
structures and histological microstructure of a tissue. This report introduces the tissue microstructures enhanced by NBI
and discusses the possibility of optimized illumination wavelength in observing living tissues.
Diffuse reflectance spectroscopy of pre- and post-treated oral submucous fibrosis: an in vivo study
Show abstract
Oral submucous fibrosis (OSF) is a high risk precancerous condition characterized by changes in the connective
tissue fibers of the lamina propria and deeper parts leading to stiffness of the mucosa and restricted mouth opening,
fibrosis of the lining mucosa of the upper digestive tract involving the oral cavity, oro- and hypo-pharynx and the upper
two-thirds of the oesophagus. Optical reflectance measurements have been used to extract diagnostic information from a
variety of tissue types, in vivo. We apply diffuse reflectance spectroscopy to quantitatively monitor tumour response to
chemotherapy. Twenty patients with submucous fibrosis were diagnosed with diffuse reflectance spectroscopy and
treated with the chemotherapy drug, Dexamethasone sodium phosphate and Hyaluronidase injection for seven weeks and
after the treatment they were again subjected to the diffuse reflectance spectroscopy. The major observed spectral
alterations on pre and post treated submucous fibrosis is an increase in the diffuse reflectance from 450 to 600 nm.
Normal mucosa has showed higher reflectance when compared to the pre and post-treated cases. The spectral changes
were quantified and correlated to conventional diagnostic results viz., maximum mouth opening, tongue protrusion and
burning sensation. The results of this study suggest that the diffuse reflectance spectroscopy may also be considered as
complementary optical techniques to monitor oral tissue transformation.
Biophotonic Approach for Disease and Virus Detection
Photonic approach to the selective inactivation of viruses with a near-infrared ultrashort pulsed laser
Show abstract
We report a photonic approach for selective inactivation of viruses with a near-infrared ultrashort pulsed
(USP) laser. We demonstrate that this method can selectively inactivate viral particles ranging from nonpathogenic
viruses such as M13 bacteriophage, tobacco mosaic virus (TMV) to pathogenic viruses like
human papillomavirus (HPV) and human immunodeficiency virus (HIV). At the same time sensitive
materials like human Jurkat T cells, human red blood cells, and mouse dendritic cells remain unharmed.
Our photonic approach could be used for the disinfection of viral pathogens in blood products and for the
treatment of blood-borne viral diseases in the clinic.
The importance of optical methods for non-invasive measurements in the skin care industry
Show abstract
Pharmaceutical and cosmetic industries are concerned with treating skin disease, as well as maintaining and promoting
skin health. They are dealing with a unique tissue that defines our body in space. As such, skin provides not only the
natural boundary with the environment inhibiting body dehydration as well as penetration of exogenous aggressors to the
body, it is also ideally situated for optical measurements. A plurality of spectroscopic and imaging methods is being
used to understand skin physiology and pathology and document the effects of topically applied products on the skin.
The obvious advantage of such methods over traditional biopsy techniques is the ability to measure the cutaneous tissue
in vivo and non-invasively. In this work, we will review such applications of various spectroscopy and imaging methods
in skin research that is of interest the cosmetic and pharmaceutical industry. Examples will be given on the importance
of optical techniques in acquiring new insights about acne pathogenesis and infant skin development.
Differentiation of normal and cancerous lung tissues by multiphoton imaging
Show abstract
In this work, we utilized multiphoton microscopy for the label-free diagnosis of non-cancerous, lung adenocarcinoma
(LAC), and lung squamous cell carcinoma (SCC) tissues from human. Our results show that the combination of second
harmonic generation (SHG) and multiphoton excited autofluorescence (MAF) signals may be used to acquire
morphological and quantitative information in discriminating cancerous from non-cancerous lung tissues. Specifically,
non-cancerous lung tissues are largely fibrotic in structure while cancerous specimens are composed primarily of tumor
masses.
Quantitative ratiometric analysis using MAF to SHG index (MAFSI or SAAID) shows that the average MAFSI for noncancerous
and LAC lung tissue pairs are 0.55 ±0.23 and 0.87±0.15 respectively. In comparison, the MAFSIs for the noncancerous
and SCC tissue pairs are 0.50±0.12 and 0.72±0.13 respectively. Intrinsic fluorescence ratio (FAD/NADH) of
SCC and non-cancerous tissues are 0.40±0.05 and 0.53±0.05 respectively, the redox ratio of SCC diminishes
significantly, indicating that increased cellular metabolic activity.
Our study shows that nonlinear optical microscopy can assist in differentiating and diagnosing pulmonary cancer from
non-cancerous tissues. With additional development, multiphoton microscopy may be used for the clinical diagnosis of
lung cancers.
Poster Session
Healing and evaluating guinea pig skin incision after surgical suture and laser tissue by welding using in vivo Raman spectroscopy
Show abstract
Changes in collagen in the wound during the healing process of guinea pig skin following surgical incisions and LTW
was evaluated using in vivo, using Raman spectroscopy. Raman spectroscopy provided information regarding the
internal structure of the proteins. After the incisions were closed either by suturing or by LTW the ratio of the Raman
peaks of the amide III (1247 cm-1) band to a peak at 1326 cm-1 used to evaluate the progression of collagen deposition.
Histopathology was used as the gold standard. LTW skin demonstrated better healing than sutured skin, exhibiting
minimal hyperkeratosis, minimal collagen deposition, near-normal surface contour, and minimal loss of dermal
appendages. This work is important to plastic surgery.
Changes of collagen, elastin, and tryptophan contents in laser welded porcine aorta tissues studied using fluorescence spectroscopy
Show abstract
The emission spectra from welded and un-welded (normal) porcine aorta tissues were measured on both sides of intima
and adventitia layers. A tunable Forsterite laser and a Cr4+: YAG laser with wavelengths of 1250nm, 1455nm and
1460nm were used to weld porcine aorta tissues.
Three emission bands emitted from three key fluorophores were studied under different welding and excitation
conditions. With excitation wavelength of 340nm, the 395nm band is associated with the emission from the structural
proteins of collagen type III and type I. The 445nm band obtained is associated with the emission of the structural
protein of elastin. The 350nm band recorded with excitation wavelength of 300nm is associated with the amino acid of
tryptophan. The relative emission intensities of collagen, elastin and tryptophan at their fluorescence peaks changes with
laser tissue welding wavelengths indicate the change of contents of those tissue molecules.
The ratio of emission peak intensities of collagen to elastin with welding laser wavelength of 1250nm increases by 0.13
as compared to the normal aorta tissue at the intimal side. For the adventitial side of aorta tissue, this ratio decreases by
0.38 in comparison with the normal tissue. These results indicate that content of collagen changes relative to elastin due
to laser tissue welding. The peak fluorescence intensity of tryptophan for both sides of welded tunica intima and
adventitia increases significantly in comparison with the normal tissue when the optimum laser welding wavelength of
1455 nm was used.
Optical birefringence of aorta tissues
Show abstract
The optical birefringence of porcine aortic tissues including heated and non-heated
tissues was studied using polarization technique. The measurements show that a whole
piece of aortic tissue has birefringence properties like a uniaxial crystal. The experiment
results indicate that the birefringence status of tissue have a potential application for
monitoring changes of tissue structure due to burning, plastic surgery, laser tissue welding
and wound healing.
Development of optical mammography based on analysis of time-resolved photon path distribution
Show abstract
A diffused optical mammography composed of a 48-channel time-resolved spectroscopy system is being developed for
breast cancer diagnosis. The system utilizes the time-correlated single photon counting method, and the detector
modules and the signal processing circuits were custom made to obtain a high signal to noise ratio and high temperature
stability with a high temporal resolution. Pulsed light generated by a Ti:Sapphire laser was irradiated to the breast, and
the transmitted light was collected by optical fibers placed on the surface of a hemispherical gantry filled with an optical
matching fluid. To reconstruct a 3D image of the breast, we employed a method using time-resolved photon path
distribution (time-resolved PPD) based on the assumption that scattering and absorption are independent of each other.
As it is not necessary to recalculate the time-resolved PPD corresponding to any changes in the absorption, we can obtain
the reconstructed image quickly. The clinical research was started in January 2007. In a comparative study with
conventional modalities, the breast cancers were detected as optically higher absorption regions. Moreover, it was
suggested that the optical mammography is useful in monitoring the effect of chemotherapy.
Application of NIR fluorescent markers to quantify expression level of HER2 receptors in carcinomas in vivo
Show abstract
HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients.
However, quantitative estimates of this important characteristic have been limited to ex vivo ELISA essays of tissue
biopsies and/or PET. We develop a novel approach in optical imaging, involving specific probes, not interfering
with the binding of the therapeutic agents, thus, excluding competition between therapy and imaging. Affibody-based
molecular probes seem to be ideal for in vivo analysis of HER2 receptors using near-infrared optical imaging.
Fluorescence intensity distributions, originating from specific markers in the tumor area, can reveal the
corresponding fluorophore concentration. We use temporal changes of the signal from a contrast agent, conjugated
with HER2-specific Affibody as a signature to monitor in vivo the receptors status in mice with different HER2
over-expressed tumor models. Kinetic model, incorporating saturation of the bound ligands in the tumor area due to
HER2 receptor concentration, is suggested to analyze relationship between tumor cell characteristics, i.e., HER2
overexpression, obtained by traditional ("golden standard") ex vivo methods (ELISA), and parameters, estimated
from the series of images in vivo. Observed correlation between these parameters and HER2 overexpression
substantiates application of our approach to quantify HER2 concentration in vivo.