This PDF file contains the front matter associated with SPIE Proceedings Volume 7895, including the Title Page, Copyright information, Table of Contents, and the Conference Committee listing.

The potential of autofluorescence microscopy under ultraviolet excitation is investigated as a method to visualize superficial epithelial microstructures and their modification with progression of disease. This method does not require the use of contrast agents, sectioning methods, or tissue preparation. Imaging of esophagus tissue is the focus of this study and deals with three main issues: a) What is the origin of the signal; b) How the gradual microstructure modification associated with various stages of esophageal disease is visualized; c) What are the designing parameters for in vivo implementation.

Extrusion or protrusion of an intervertebral disc is a common, frequently debilitating, painful, and sometimes fatal neurologic disease in the chondrodystrophic dog (dachshund, Pekingese, etc.). A similar condition of intervertebral disc degeneration with extrusion/protrusion is also a relatively common neurologic condition in human patients. Degeneration of the relatively avascular chondrodystrophoid intervertebral disc is associated with loss of water content, increased collagen, and deposits of calcified mineral in the nucleus pulposus. Current diagnostic methods have many limitations for providing accurate information regarding disc composition in situ prior to surgical intervention. Disc composition (i.e., mineralization), can influence the type of treatment regime and potentially prognosis and recurrence rates. The objective of this study is to investigate the feasibility of using a fiber-needle spectroscopy sensor to analyze the changes of tissue compositions involved in the chondrodystrophoid condition of the canine intervertebral disc. The nucleous pulposus, in which the metaplastic process / degeneration develops, is approximately 2mm thick and 5mm in diameter in the dachshund-sized dog. It lies in the center of the disc, surrounded by the annulus fibrosis and is enclosed by cartilaginous vertebral endplates cranially and caudally. This "shallow-and-small-slab" geometry limits the configuration of a fiber probe to sense the disc tissue volume without interference from the vertebrae. A single-fiber sensor is inserted into a 20 gauge myelographic spinal needle for insertion into the disc in situ and connected via a bifurcated fiber to the light source and a spectrometer. A tungsten light source and a 940nm light-emitting-diode are combined for spectral illumination covering VIS/NIR with expected improved sensitivity to water. Analysis of the reflectance spectra is expected to provide information of scattering and absorption compositions of tissue in proximity to the fiber-tip. Preliminary measurements on cadaveric canine intervertebral discs indicated significant reduction of scattering constituents and possible diminishment of water content after percutaneous laser disc ablation (PLDA). This fiber-needle based sensing configuration may be feasible for integrating the evaluation of calcification and water content into the work-flow of holmium:YAG laser disc ablation for pre-operative in-line detection and post-operative evaluation of therapeutic interventions regarding the chondrodystrophic disc.

In this work, we applied multi-photon microscopy to image and characterize the extent of tissue glycation. Specifically, features of multi-photon autofluorescence (MPAF) and second harmonic generation (SHG) images were presented. Our study shows that multiphoton imaging is capable of providing qualitative and quantitative information of the extent of tissue glycation in ribose treated samples including bovine sclera and vessel and that this approach may be useful for clinical evaluation of advanced glycation endproducts (AGEs) formation.

In this paper we present the results of a study to distinguish cervical cancer patients [ N=50] from healthy subjects [N=50] based on the Fluorescence Emission Spectra [FES] and Stokes' Shift Spectra [SSS] of blood and urine. FES was obtained from the cellular fraction of blood and urine by excitation at 400 nm. SSS was obtained from blood plasma and urine with Δλ of 70nm. In the FES of blood cellular fraction, the ratio of intensity of the two bands due to neutral porphyrin and basic porphyrin [I630 / I580] was 1 for normal controls and 3 for cervical cancers. In the SSS of plasma, the average ratio of intensity of the two bands due to tryptophan and collagen [I305 nm / I340 nm] was 1.9 for normal controls, 1.1 for early cervical cancers and 0.9 for advanced cervical cancers In the SSS of urine, the ratio of intensity of the two bands due to flavin and NADH [I450 nm / I360 nm] was 0.2 for normal controls and 0.8 for cancer patients. A discriminant analysis combining all three parameters showed a sensitivity of 80% and specificity of 78% for this technique. In this study we show that fluorescence spectroscopy of blood and urine could develop into a promising technique for non-invasive diagnosis and screening of cervical cancers and would appropriately supplement or complement currently used techniques.

Time-resolved spectroscopy and near infrared imaging enhanced by receptor-targeted contrast agents for prostate cancer detection will be presented. Two contrast agents, Cybesin and Cytate, were investigated using time-resolved spectroscopy in aqueous solution and cancerous and normal prostate tissues. The time evolution of the fluorescent dipole in solution was studied using a system of first-order linear differential equations containing two main parameters: the decay rate of emission and the rate of one orthogonal emission component transferring to another. An analytical polarization model was developed and used to extract rotational times and fluorescence anisotropies of the contrast agents in prostate tissues. The differences of rotational times and polarization anisotropies were observed for Cybesin (Cytate) in cancerous and normal prostate tissue, which reflect preferred bond of contrast agents and cancerous tissue cells. The conjugation of Cybesin (Cytate) to prostate cancerous cells offers high contrast between normal and cancerous tissues.

Time domain diffuse optical spectroscopy provides the absorption and scattering properties of biological tissues and diffusive materials. Few measurements are available at discrete wavelengths beyond 1100 nm, and just one time-domain system continuously tuneable up to 1400 nm. We developed a time-domain system, based on a continuously tuneable supercontinuum pulsed source, and a custom InGaAs/InP Single-Photon Avalanche Diode. Operation was demonstrated in the 1100-1700 nm range with a spectral resolution of 15 nm, a temporal resolution of 150 ps and a background of 6000 counts/s. A first example of application on the optical characterization of collagen powder is given.

In this paper we show the results of multi-spectral Mueller Imaging applied to the analysis of human colon cancer in a backscattering configuration with diffuse light illumination. The analyzed sample behaves as a pure depolarizer. The depolarization power, for both healthy and cancerous zones, is lower for linearly than for circularly polarized incident light for all used wavelengths and increases with increasing wavelength. Based on their visual staging and polarimetric responses, we chose specific zones which we correlated to the histology of the corresponding cuts. The histological examination shows that we see a multilayer interaction in both healthy and abnormal zones, if the light penetration depth is sufficient. The measured depolarization depends on several factors: the presence or absence of tumor, the microscopic structure of cancer (ratio between cellular density and stroma), its exophytic (budding) or endophytic (penetrating) nature, its thickness, the degree of cancer penetration in deeper layers and the nature of healthy tissue left under abnormal layers. These results demonstrate that multi-spectral Mueller imaging can provide useful contrasts for the quick staging of human colon cancer ex-vivo, with additional information about cancerous zones with different microscopic structures.

In this study the diagnostic potential of synchronous luminescence spectroscopy (SLS) technique for the characterization of normal and different pathological condition of cervix viz., moderately differentiated squamous cell carcinoma (MDSCC), poorly differentiated squamous cell carcinoma (PDSCC) and well differentiated squamous cell carcinoma (WDSSC). Synchronous fluorescence spectra were measured for 70 abnormal cases and 30 normal subjects. Characteristic, highly resolved peaks and significant spectral differences between normal and MDSCC, PDSCC and WDSCC cervical blood formed elements were obtained. The synchronous luminescence spectra of formed elements of normal and abnormal cervical cancer patients were subjected to statistical analysis. Synchronous luminescence spectroscopy provides 90% sensitivity and 92.6% specificity.

We present the application of two fibre-optic-coupled time-resolved spectrofluorometers and a compact steady-state diffuse reflected light/fluorescence spectrometer to in vivo and ex vivo studies of skin cancer and osteoarthritis. In a clinical study of skin cancer, 27 lesions on 25 patients were investigated in vivo before surgical excision of the region measured. Preliminary analysis reveals a statistically significant decrease in the autofluorescence lifetime of basal cell carcinomas compared to neighbouring healthy tissue. A study of autofluorescence signals associated with the onset of osteoarthritis indicates autofluorescence lifetime changes associated with collagen degradation.

Stokes Shift Spectroscopy (SSS) has emerged as a promising modality in the discrimination of normal from different pathological prostate tissues. Stokes shift (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 prostate tissues were observed. The SS spectra of normal, hyperplasia and malignant tissues shows the distinct peaks around 300, 345, 440 and 510 nm is attributed to tryptophan, collagen, NADH and flavin respectively. To quantify the spectral differences between normal and different pathological prostate tissues are verified by statistical analysis.

Needle-based core-biopsy to locate prostate cancer relies heavily upon trans-rectal ultrasound (TRUS) imaging guidance. Ultrasonographic findings of classic hypoechoic peripheral zone lesions have a low specificity of ~28%, a low positive predictive value of ~29%, and an overall accuracy of ~43%, in prostate cancer diagnosis. The prevalence of isoechoic or nearly invisible prostate cancers on ultrasonography ranges from 25 to 42%. As a result, TRUS is useful and convenient to direct the needle trajectory following a systematic biopsy sampling template rather than to target only the potentially malignant lesion for focal-biopsy. To address this deficiency in the first-line of prostate cancer imaging, a trans-rectal ultrasound-coupled spectral tomography (TRUST) approach is being developed to non-invasively resolve the likely optical signatures of prostate malignancy. The approach has evolved from using one NIR wavelength to two NIR bands, and recently to three bands of NIR spectrum information. The concept has been evaluated on one normal canine prostate and three dogs with implanted prostate tumor developed as a model. The initial results implementing TRUST on the canine prostate tumor model includes: (1) quantifying substantially increased total hemoglobin concentration over the time-course of imaging in a rapidly growing prostate tumor; (2) confirming hypoxia in a prostatic cystic lesion; and (3) imaging hypoxic changes of a necrotic prostate tumor. Despite these interesting results, intensive technologic development is necessary for translating the approach to benefiting clinical practice, wherein the ultimate utility is not possibly to eliminate needle-biopsy but to perform focal-biopsy that is only necessary to confirm the cancer, as well as to monitor and predict treatment responses.

In surgical treatment of pancreatic cancers, the effectiveness of the procedures largely depends on the ability to completely and precisely remove the malignant tumors. We present the ex-vivo use of oblique incidence diffuse reflectance spectroscopy (OIRDS) to detect and differentiate normal from neoplastic tissue. An OIRDS probe has been constructed to provide scattering and absorption information of the pancreatic tissue. To reveal the physiological origin of the difference in these optical signatures, the optical scattering coefficients were extracted along the pancreatic duct with 1-cm spacing. Experimental results show that OIDRS was able to successfully determinate the tumor margins based on the higher optical scattering on malignant tissue.

Three-dimensional (3D) localization of human cancerous prostate tissue embedded in normal prostate tissue was demonstrated and characterized using backscattering scanning polarization imaging and independent component analysis (ICA). Backscattering two-dimensional (2D) images of a tissue sample illuminated with a raster scanning laser beam were recorded using a CCD camera to obtain multiple angular views of the target embedded inside the tissue medium. The 3D location of the object was retrieved by matching the leading Independent Component(s) of intensity distribution of images to the numerical propagation of the target to the surface of the tissue medium.

The metabolic coenzymes NADH and FAD are autofluorescent and can be monitored non-destructively and without exogenous labels, using optical techniques. These endogenous fluorophores which are present in the cells and tissues gives rise to different fluorescence emission/excitation spectra between the normal and different diseased conditions. In the resent years, finding the optical redox ratio i.e., the ratio of the fluorescence intensity of FAD and NADH, gives the relative change in the oxidation-reduction state of the cells. Unlike other organs oral cavity has lined with variety of mucosal types. We investigated in vivo Optical redox ratio for four different anatomical locations viz., cheek mucosa, vermilion border of the lip, Hard palate, dorsal side of the tongue of healthy oral cavity. We measured this ratio for 20 healthy subjects and the redox ratio was significantly different between the different anatomical locations. The statistical significance was also investigated.

The protocol for prostate cancer diagnosis, currently based on ultrasound guided biopsy, is limited by a lack of relevance. To improve this protocol, a new approach was proposed combining optical and ultrasound measurements to guide biopsy specifically to the tumors. Adding an optical measurement modality into an already existing ultrasound probe is challenging as the overall size of the system should not exceed a given dimension so as to fit the operative environment. Moreover, examination should not take more than 15 min to avoid any complication. A combined ultrasound and optical endorectal probe was designed to comply with the constraints of the sterilization protocols, the examination duration and required compactness. Therefore a totally innovative pulsed laser source has been designed to meet compactness requirements while providing accurate time-resolved measurements. A dedicated multi-channel photon counting system was optimized to decrease the examination duration. A fast reconstruction method based on the analysis of the intensity and time of flight of the detected photons has been associated to provide 3D localization of fluorescent dots almost immediately after acquisition. The bi-modal probe was capable of withstanding the sterilization procedures. The performance of the compact laser source has been shown at the same level as that of a standard laboratory Titane:Sapphire laser. The dedicated photon counting solution was capable of acquiring optical data in less than one minute. To evaluate the overall performance of the system in dealing with a realistic background signal, measurements and reconstructions were conducted on prostate mimicking phantom and in vivo.

The management of modern traumatic war wounds remains a significant challenge for clinicians. This is a reflection of the extensive osseous and soft-tissue damage caused by blasts and high-energy projectiles. The ensuing inflammatory response ultimately dictates the pace of wound healing and tissue regeneration. Consequently, the eventual timing of wound closure or definitive coverage is often subjectively based. Some wounds require an extended period of time to close or fail to remain closed, despite the use and application of novel wound-specific treatment modalities. Aside from impaired wound healing, additional wound complications include wound infection, biofilm formation, and heterotopic ossification (the pathological mineralization of soft tissues). An understanding of the molecular environment of acute wounds throughout the debridement process can provide valuable insight into the mechanisms associated with the eventual wound outcome. The analysis of Raman spectra of ex vivo wound biopsy tissue obtained from serial traumatic wound debridements reveals a decreased 1665 cm-1/1445 cm-1 band area ratio in impaired healing wounds, indicative of an impaired remodeling process, in addition to a decreased 1240 cm-1/1270cm-1. The examination of debrided tissue exhibits mineralization during the early development of heterotopic ossification. Finally, preliminary results suggest that Fourier transform infrared (FT-IR) images of wound effluent may be able to provide early microbiological information about the wound.

An objective method to measure the effectiveness of regional anesthesia can reduce time and unintended pain inflicted to the patient. A prospective observational study was performed on 22 patients during a local anesthesia before undergoing hand surgery. Two non-invasive techniques thermal and oxygenation imaging were applied to observe the region affected by the peripheral block and the results were compared to the standard cold sensation test. The supraclavicular block was placed under ultrasound guidance around the brachial plexus by injecting 20 cc Ropivacaine. The sedation causes a relaxation of the muscles around the blood vessels resulting in dilatation and hence an increase of blood perfusion, skin temperature and skin oxygenation in the lower arm and hand. Temperatures were acquired with an IR thermal camera (FLIR ThermoCam SC640). The data were recorded and analyzed with the ThermaCam^TMResearcher and Matlab software. Narrow band spectral images were acquired at selected wavelengths with a CCD camera either combined with a Liquid Crystal Tunable Filter (420-730 nm) or a tunable hyper-wavelength LED light source (450-880nm). Concentration changes of oxygenated and deoxygenated hemoglobin in the dermis of the skin were calculated using the modified Lambert Beer equation. Both imaging methods showed distinct oxygenation and temperature differences at the surface of the skin of the hand with a good correlation to the anesthetized areas. A temperature response was visible within 5 minutes compared to the standard of 30 minutes. Both non-contact methods show to be more objective and can have an earlier prediction for the effectiveness of the anesthetic block.

We report inactivation of encephalomyocarditis virus and salmonella typhimurium by a visible femtosecond laser. Our results show that inactivation of virus and bacterium by an ultrashort pulsed laser light might involve completely different mechanisms. Inactivation of viruses by an ultrashort pulsed laser might involve disruption of their protein coat through laser-induced excitation of large-amplitude acoustic vibrations. On the other hand, inactivation of bacteria is most likely related to the disruption of their metabolism by the DNA relaxation process caused by irradiation of ultrashort pulsed lasers.

A miniaturized scanning mechanism is a crucial component in the creation of endoscopes for microscopic imaging. Several groups have developed resonant scanners (e.g., spiral or Lissajous scan pattern), but these suffer from limitations in non-uniform spatial coverage and sampling time, in comparison to a raster scanner. Additionally, a resonant scanner lacks the ability to perform line-scan imaging, a crucial capability in measuring a variety of fast, dynamic physiological phenomena (e.g., blood flow, molecular diffusion, etc.). However, current miniaturized raster scanners are limited in terms of their physical dimensions and scan speed. We demonstrate a novel hybrid resonant/non-resonant miniaturized raster scanner, fabricated by mounting a double clad fiber onto two perpendicularly oriented piezo bimorphs. The fiber scanner has a total length of 2.6cm, a width/thickness [less than or equal to] 1mm, achieves [greater than or equal to] fiber tip deflection for both the resonant and non-resonant axes, and allows for imaging at approximately 4 frames per second (512 lines per frame). An essentially uniform spatial coverage and sampling time can be achieved by utilizing the middle portion (e.g., middle 500 μm) of the resonant scanning range. The small size allows for the fiber scanner to be easily packaged along with miniaturized lenses to form an endoscope for microscopic imaging. We bonded a stiffening fiber alongside the vibrating fiber to break its cylindrical symmetry. Thus, only one vibration mode is excited, generating a purely linear spatial motion. In order to demonstrate the fiber scanner's imaging capabilities we have taken transmission and fluorescence images, in which the double clad fiber's inner clad is used for fluorescence collection.

The microinjection of organelles, plants, particles or chemical solutions into Amoeba proteus coupled with spectroscopic analysis and observed for a period of time provides a unique new model for cancer treatment and studies. The amoeba is a eukaryote having many similar features of mammalian cells. The amoeba biochemical functions monitored spectroscopically can provide time sequence in vivo information about many metabolic transitions and metabolic exchanges between cellar organelles and substances microinjected into the amoeba. It is possible to microinject algae, plant mitochondria, drugs or carcinogenic solutions followed by recording the native fluorescence spectra of these composites. This model can be used to spectroscopically monitor the pre-metabolic transitions in developing diseased cells such as a cancer. Knowing specific metabolic transitions could offer solutions to inhibit cancer or reverse it as well as many other diseases. In the present study a simple experiment was designed to test the feasibility of this unique new model by injecting algae and chloroplasts into amoeba. The nonradiative dynamics found from these composites are evidence in terms of the emission ratios between the intensities at 337nm and 419nm; and 684nm bands. There were reductions in the metabolic and photosynthetic processes in amoebae that were microinjected with chloroplasts and zoochlorellae as well of those amoebae that ingested the algae and chloroplasts. The changes in the intensity of the emissions of the peaks indicate that the zoochlorellae lived in the amoebae for ten days. Spectral changes in intensity under the UV and 633nm wavelength excitation are from the energy transfer of DNA and RNA, protein-bound chromophores and chlorophylls present in zoochlorellae undergoing photosynthesis. The fluorescence spectroscopic probes established the biochemical interplay between the cell organelles and the algae present in the cell cytoplasm. This hybrid state is indicative that a symbiotic system is in place and the results definitely support the potential use of this unique new model. This model many help in plant / animal and cancer processes.