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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.