This PDF file contains the front matter associated with SPIE Proceedings Volume 9703, including the Title Page, Copyright information, Table of Contents, Introduction, and the Conference Committee listing.

We are establishing a new paradigm in mid-infrared molecular sensing, mapping and imaging to open up the midinfrared spectral region for in vivo (i.e. in person) medical diagnostics and surgery. Thus, we are working towards the mid-infrared optical biopsy (‘opsy’ look at, bio the biology) in situ in the body for real-time diagnosis. This new paradigm will be enabled through focused development of devices and systems which are robust, functionally designed, safe, compact and cost effective and are based on active and passive mid-infrared optical fibers. In particular, this will enable early diagnosis of external cancers, mid-infrared detection of cancer-margins during external surgery for precise removal of diseased tissue, in one go during the surgery, and mid-infrared endoscopy for early diagnosis of internal cancers and their precision removal. The mid-infrared spectral region has previously lacked portable, bright sources. We set a record in demonstrating extreme broad-band supercontinuum generated light 1.4 to 13.3 microns in a specially engineered, high numerical aperture mid-infrared optical fiber. The active mid-infrared fiber broadband supercontinuum for the first time offers the possibility of a bright mid-infrared wideband source in a portable package as a first step for medical fiber-based systems operating in the mid-infrared. Moreover, mid-infrared molecular mapping and imaging is potentially a disruptive technology to give improved monitoring of the environment, energy efficiency, security, agriculture and in manufacturing and chemical processing. This work is in part supported by the European Commission: Framework Seven (FP7) Large-Scale Integrated Project MINERVA: MId-to-NEaR- infrared spectroscopy for improVed medical diAgnostics (317803; www.minerva-project.eu).

Pathologists find it notoriously difficult to provide both inter- and intra-observer agreement on a diagnosis of early gastrointestinal cancers. Vibrational spectroscopic approaches have shown their value in providing molecular compositional data from tissue samples and therefore enabling the identification of disease specific changes, when combined with multivariate techniques. Mid-infrared microscopic imaging is undergoing rapid developments in sources, detectors and spectrometers. Here we explore the use of high magnification FTIR for GI cancers and consider how the MINERVA (MId- to NEaR infrared spectroscopy for improVed medical diAgnostics) project, which is developing discrete frequency IR imaging tools will enable histopathologists to obtain rapid molecular images form unstained tissue sections.

The extension of supercontinuum (SC) sources into the mid-infrared, via the use of uoride and chalcogenide optical fibers, potentially offers the high radiance of a laser combined with spectral coverage far exceeding that of typical tunable lasers and comparable to traditional black-body emitters. Together with advances in mid-IR imaging detectors and novel tunable filter designs, such supercontinua hold considerable potential as sources of illumination for spectrally-resolved microscopy targeting applications such as rapid histological screening. The ability to rapidly and arbitrarily select particular wavelengths of interest from a broad emission spectrum, covering a wide range of biologically relevant targets, lends itself to image acquisition only at key relevant wavelengths leading to more manageable datasets. However, in addition to offering new imaging modalities, SC sources also present a range of challenges to successful integration with typical spectral microscopy instrumentation, including appropriate utilisation of their high spatial coherence. In this paper the application of SC sources to spectrally-resolved microscopy in the mid-IR is discussed and systems-integration considerations specific to these sources highlighted. Preliminary results in the 3-5μm region, obtained within the European FP7 project MINERVA, are also presented here.

FTIR spectroscopy is an emerging technology with high potential for cancer diagnosis but with particular physical phenomena that require special processing. Little work has been done in the field with the aim of registering hyperspectral Fourier-Transform Infrared (FTIR) spectroscopic images and Hematoxilin and Eosin (HE) stained histological images of contiguous slices of tissue. This registration is necessary to transfer the location of relevant structures that the pathologist may identify in the gold standard HE images. A two-step registration framework is presented where a representative gray image extracted from the FTIR hypercube is used as an input. This representative image, which must have a spatial contrast as similar as possible to a gray image obtained from the HE image, is calculated through the spectrum variation in the fingerprint region. In the first step of the registration algorithm a similarity transformation is estimated from interest points, which are automatically detected by the popular SURF algorithm. In the second stage, a variational registration framework defined in the frequency domain compensates for local anatomical variations between both images. After a proper tuning of some parameters the proposed registration framework works in an automated way. The method was tested on 7 samples of colon tissue in different stages of cancer. Very promising qualitative and quantitative results were obtained (a mean correlation ratio of 92.16% with a standard deviation of 3.10%).

FTIR is a well-established technique and there is significant interest in applying this technique to medical diagnostics e.g. to detect cancer. The introduction of focal plane array (FPA) detectors means that FTIR is particularly suited to rapid imaging of biopsy sections as an adjunct to digital pathology. Until recently however each pixel in the image has been limited to a minimum of 5.5 µm which results in a comparatively low magnification image or histology applications and potentially the loss of important diagnostic information. The recent introduction of higher magnification optics gives image pixels that cover approx. 1.1 µm. This reduction in image pixel size gives images of higher magnification and improved spatial detail can be observed. However, the effect of increasing the magnification on spectral quality and the ability to discriminate between disease states is not well studied. In this work we test the discriminatory performance of FTIR imaging using both standard (5.5 µm) and high (1.1 µm) magnification for the detection of colorectal cancer and explore the effect of binning to degrade high resolution images to determine whether similar diagnostic information and performance can be obtained using both magnifications. Results indicate that diagnostic performance using high magnification may be reduced as compared to standard magnification when using existing multivariate approaches. Reduction of the high magnification data to standard magnification via binning can potentially recover some of the lost performance.

The rapid inspection of suspicious skin lesions for pathological cell types is the objective of optical point of care diagnostics technologies. A marker free fast diagnosis of skin malignancies would overcome the limitations of the current gold standard surgical biopsy. The time consuming and costly biopsy procedure requires the inspection of each sample by a trained pathologist, which limits the analysis of potentially malignant lesions. Optical technologies like RAMAN or infrared spectroscopy, which provide both, localization and chemical information, can be used to differentiate malignant from healthy tissue by the analysis of multi cell structures and cell type specific spectra. We here report the application of midIR spectroscopy towards fast and reliable skin diagnostics. Within the European research project MINERVA we developed standardized in vitro skin systems with increasing complexity, from single skin cell types as fibroblasts, keratinocytes and melanoma cells, to mixtures of these and finally three dimensional human skin equivalents. The standards were characterized in the established midIR range and also with newly developed systems for fast imaging up to 12 μm. The analysis of the spectra by novel data processing algorithms demonstrated the clear separation of all cell types, especially the tumor cells. The signals from single cell layers were sufficient for cell type differentiation. We have compared different midIR systems and found all of them suitable for specific cell type identification. Our data demonstrate the potential of midIR spectroscopy for fast image acquisition and an improved data processing as sensitive and specific optical biopsy technology.

We developed crystalline AgClBr fibers of diameters 0.7-0.9mm that are flexible, non-toxic, insoluble in water and highly transparent between 4-15µm. We used these fibers for various sensing applications. Highly sensitive absorption measurements in the mid-IR may be carried out by Fiber-optic Evanescent Wave Spectroscopy (FEWS). A typical FEWS system is based on three mid-IR components: a tunable source, a detector and a AgClBr fiber sensor that is brought in contact with the samples. We used FTIR spectrometers or tunable gas lasers or quantum cascade lasers (QCLs) as mid-IR sources. We used this FEWS system for measurements on gases, liquids and solids. In particular we used it for several biomedical applications. Measurements in vivo: (1) Early detection of skin diseases (e.g. melanoma). (2) Measurements on cells and bacteria. (3) Measurements on cornea. Measurements in vitro: (4) Characterization of urinary and biliary stones. (5) Blood measurements. The FEWS method is simple, inexpensive and does not require sample processing. It would be useful for diagnostic measurements on the outer part of the body of a patient, as well as for endoscopic measurements. It would also useful for measurements on tissue samples removed from the body. In addition we develop Scanning Near-field Infrared Microscope that will be used for spectral imaging with sub-wavelength resolution in the mid-IR. The various AgClBr fiber-optic sensors are expected to be important diagnostic tools at the hand of physicians in the future.

In the UK, it is now recognised that 1 in 2 people born after 1960 will develop some form of cancer during their lifetime. Diagnosing patients whilst in the early stages drastically improves their chances of survival but up until now the gold standard for cancer detection is via a lengthy excision biopsy procedure, which relies on the skill of a histopathologist. Evidently, the need for a faster solution is paramount. The mid-infrared (MIR) spectral region covers the wavelengths 3-25 μm and characteristic vibrational spectra unique to each molecular type. Subtle changes in the specific spectral response within this region are indicative of changes within the cells relative to normal cells, signifying the presence or absence of a disease. Our goal is to carry out disease diagnosis in vivo. Reaching these wavelengths has previously presented difficulties as conventional MIR blackbody light sources are weak and optical fibers for transmitting MIR light to/from tissue in vivo can be limited by strong material absorption such as silica glass >2.4 μm and tellurite, and heavy metal fluoride, >4.75 μm. However, chalcogenide glasses have been shown to transmit MIR light out to 25 μm. This paper reports on a glass composition in the Ge-Sb-Se system and its suitability as an optical fiber for the transmission of MIR to and from tissue samples, enabling in vivo mapping for an immediate diagnostic response- a technique termed ‘optical biopsy’.

We report on an experimental demonstration of mid-infrared cascaded supercontinuum generation in commercial silica, fluoride, and chalcogenide fibers as a potentially cheap and practical alternative to direct pumping schemes. A pump continuum up to 4.4 μm was generated in cascaded silica and fluoride fibers by an amplified 1.55 μm nanosecond diode laser. By pumping a commercial Ge₁₀As₂₂Se₆₈ single-material photonic crystal fiber with 135.7 mW of the pump continuum from 3.5- 4.4 μm, we obtained a continuum up to 7.2 μm with a total output power after the collimating lens of 54.5 mW, and 3.7 mW above 4.5 μm.

spectroscopy has until now been greatly limited by the availability of lightsources. The choice has generally stood between a laser whose narrow spectrum limits flexibility or a globar, whose low brightness limits signal to noise ratio. Mid-IR supercontinuum sources, which can deliver an ultra-broad spectrum with a million times higher brightness than a globar, are now appearing to fill the performance gap between the traditional lightsources. The generation of a supercontinuum is a highly nonlinear process produced by high peak power pulses propagating through a nonlinear medium. Since the underlying processes are fundamentally random there will normally be some pulse to pulse fluctuation in the output light which can cause problems in spectroscopy. Most of the mid-IR supercontinuum sources shown to date have also been limited to pulse repetition rates of only a few tens of kilohertz which makes it difficult to apply them to the popular FTIR spectroscopy techniques.

Here we will demonstrate a fully packaged, all-fiber, turn-key, low noise, 4.8W, 1.8-4.2 μm supercontinuum source, which can operate with variable repetition rates of up to 30 MHz. In addition we will discuss ways to reduce and counter the effects of pulse fluctuations and we demonstrate optimization of the output spectrum of the source for various applications. Such a source can give any mid-IR optics lab access to a performance which has previously only been available from dedicated beamlines at huge synchrotron facilities.

Unacceptably large percentage (20-40%) of breast cancer lumpectomy patients are required to undergo multiple surgeries when positive margins are found upon post-operative histologic assessment. If the margin status can be determined during surgery, surgeon can resect additional tissues to achieve tumor-free margin, which will reduce the need for additional surgeries. Spectrally encoded confocal microscopy (SECM) is a high-speed reflectance confocal microscopy technology that has a potential to image the entire surgical margin within a short procedural time. Previously, SECM was shown to rapidly image a large area (10 mm by 10 mm) of human esophageal tissue within a short procedural time (15 seconds). When used in lumpectomy, SECM will be able to image the entire margin surface of ~30 cm2 in around 7.5 minutes. SECM images will then be used to determine margin status intra-operatively. In this paper, we present results from a study of testing accuracy of SECM for diagnosing malignant breast tissues. We have imaged freshly-excised breast specimens (N=46) with SECM. SECM images clearly visualized histomorphologic features associated with normal/benign and malignant breast tissues in a similar manner to histologic images. Diagnostic accuracy was tested by comparing SECM diagnoses made by three junior pathologists with corresponding histologic diagnoses made by a senior pathologist. SECM sensitivity and specificity were high, 0.91 and 0.93, respectively. Intra-observer agreement and inter-observer agreement were also high, 0.87 and 0.84, respectively. Results from this study showed that SECM has a potential to accurately determine margin status during breast cancer lumpectomy.

FFOCT (Full Field Optical Coherence Tomography) is a novel optical technology that gives access to very high resolution tomography images of biological tissues within minutes, non-invasively. This makes it an attractive tool to bridge the gap between medical imaging modalities (MRI, ultrasound, CT) used for cancer lesion identification or targeting and histological diagnosis. Clinical tissue specimens, such as surgical cancer margins or biopsies, can potentially be assessed rapidly, by the clinician, in the aim to help him decide on the course of action. A fast FFOCT prototype was built, that provides 1cm2 images with 1 µm resolution in 1 minute, and can accommodate samples up to 50mm diameter. Specific work was carried out to implement a large sample holder, high-speed image acquisition system, optimized scanning, and accelerated GPU tiles stitching. Results obtained on breast, urology, and digestive tissues show the efficiency of the technique for the detection of cancer on clinical tissue specimens, and reinforce the clinical relevance of the technique. The technical and clinical results show that the fast FFOCT system can successfully be used for a fast assessment of cancer excision margins or biopsies providing a very valuable tool in the clinical environment.

Intraoperative margin assessment to evaluate resected tissue margins for neoplastic tissue is performed to prevent reoperations following breast-conserving surgery. High resolution microendoscopy (HRME) can rapidly acquire images of fresh tissue specimens, but is limited by low image contrast in tissues with high optical scattering. In this study we evaluated two techniques to reduce out-of-focus light: HRME image acquisition with structured illumination (SI-HRME) and topical application of Lugol’s Iodine. Fresh breast tissue specimens from 19 patients were stained with proflavine alone or Lugol’s Iodine and proflavine. Images of tissue specimens were acquired using a confocal microscope and an HRME system with and without structured illumination. Images were evaluated based on visual and quantitative assessment of image contrast. The highest mean contrast was measured in confocal images stained with proflavine. Contrast was significantly lower in HRME images stained with proflavine; however, incorporation of structured illumination significantly increased contrast in HRME images to levels comparable to that in confocal images. The addition of Lugol’s Iodine did not increase mean contrast significantly for HRME or SI-HRME images. These findings suggest that structured illumination could potentially be used to increase contrast in HRME images of breast tissue for rapid image acquisition.

We created a line-scanning, stage scanning confocal microscope as part of a new procedure: video assisted micrographic surgery (VAMS). The need for rapid pathological assessment of the tissue on the surface of skin excisions very large since there are 3.5 million new skin cancers diagnosed annually in the United States. The new design presented here is a confocal microscope without any scanning optics. Instead, a line is focused in space and the sample, which is flattened, is physically translated such that the line scans across its face in a direction perpendicular to the line its self. The line is 6mm long and the stage is capable of scanning 50 mm, hence the field of view is quite large. The theoretical diffraction-limited resolution is 0.7um lateral and 3.7um axial. However, in this preliminary report, we present initial results that are a factor of 5-7 poorer in resolution. The results are encouraging because they demonstrate that the linear array detector measures sufficient signal from fluorescently labeled tissue and also demonstrate the large field of view achievable with VAMS.

We present a novel method, based on encoder mapping OCT imaging, for real-time guidance of core biopsy procedures. This method provides real-time feedback to the interventional radiologist, such that he/she can reorient the needle during the biopsy and sample the most representative area of the suspicious mass that is being investigated. This aspect is very important for tailoring therapy to the specific cancer based on biomarker analysis, which will become one of the next big advances in our search for the optimal cancer therapy. To enable individualized treatment, the genetic constitution and the DNA repair status in the affected areas is needed for each patient. Thus, representative sampling of the tumor is needed for analyzing various biomarkers, which are used as a tool to personalize cancer therapy. The encoder-based OCT enables samping of large size masses and provides full control on the imaging probe, which is passed through the bore of the biopsy guidance needle. The OCT image is built gradually, based on the feedback of an optical encoder which senses the incremental movement of the needle with a few microns resolution. Tissue mapping is independent of the needle speed, while it is advanced through the tissue. The OCT frame is analyzed in real-time and tissue cellularity is reported in a very simple manner (pie chart). Our preliminary study on a rabbit model of cancer has demonstrated the capability of this technology for accurately differentiating between viable cancer and heterogeneous or necrotic tissue.

Background: Routine urological surgery frequently requires rapid on-site histopathological tissue evaluation either during biopsy or intra-operative procedure. However, resected tissue needs to undergo processing, which is not only time consuming but may also create artifacts hindering real-time tissue assessment. Likewise, pathologist often relies on several ancillary methods, in addition to H&E to arrive at a definitive diagnosis. Although, helpful these techniques are tedious and time consuming and often show overlapping results. Therefore, there is a need for an imaging tool that can rapidly assess tissue in real-time at cellular level. Multiphoton microscopy (MPM) is one such technique that can generate histology-quality images from fresh and fixed tissue solely based on their intrinsic autofluorescence emission, without the need for tissue processing or staining.

Design: Fresh tissue sections (neoplastic and non-neoplastic) from biopsy and surgical specimens of bladder and kidney were obtained. Unstained deparaffinized slides from biopsy of medical kidney disease and oncocytic renal neoplasms were also obtained. MPM images were acquired using with an Olympus FluoView FV1000MPE system. After imaging, fresh tissues were submitted for routine histopathology.

Results: Based on the architectural and cellular details of the tissue, MPM could characterize normal components of bladder and kidney. Neoplastic tissue could be differentiated from non-neoplastic tissue and could be further classified as per histopathological convention. Some of the tumors had unique MPM signatures not otherwise seen on H&E sections. Various subtypes of glomerular lesions were identified as well as renal oncocytic neoplasms were differentiated on unstained deparaffinized slides.

Conclusions: We envision MPM to become an integral part of regular diagnostic workflow for rapid assessment of tissue. MPM can be used to evaluate the adequacy of biopsies and triage tissues for ancillary studies. It can also be used as an adjunct to frozen section analysis for intra-operative margin assessment. Further, it can play an important role for pathologist for guiding specimen grossing, selecting tissue for tumor banking and as a rapid ancillary diagnostic tool.

Widely used methods for preparing and viewing tissue specimens at microscopic resolution have not changed for over a century. They provide high-quality images but can involve time-frames of hours or even weeks, depending on logistics. There is increasing interest in slide-free methods for rapid tissue analysis that can both decrease turn-around times and reduce costs. One new approach is MUSE (microscopy with UV surface excitation), which exploits the shallow penetration of UV light to excite fluorescent signals from only the most superficial tissue elements. The method is non-destructive, and eliminates requirement for conventional histology processing, formalin fixation, paraffin embedding, or thin sectioning. It requires no lasers, confocal, multiphoton or optical coherence tomography optics. MUSE generates diagnostic-quality histological images that can be rendered to resemble conventional hematoxylin- and eosin-stained samples, with enhanced topographical information, from fresh or fixed, but unsectioned tissue, rapidly, with high resolution, simply and inexpensively. We anticipate that there could be widespread adoption in research facilities, hospital-based and stand-alone clinical settings, in local or regional pathology labs, as well as in low-resource environments.

We have developed an automated, wide-field optical coherence tomography (OCT)-based imaging device (OTIS^TM Perimeter Medical Imaging) for peri-operative, ex-vivo tissue imaging. This device features automated image acquisition, enabling rapid capture of high-resolution (15 μm) OCT images from samples up to 10 cm in diameter. We report on the iterative progression of device development from phantom and pre-clinical (tumor xenograft) models through to initial clinical results. We discuss the challenges associated with proving a novel imaging technology against the clinical “gold standard” of conventional post-operative pathology.

INVITED TALK Advances in imaging tissue microstructure in living subjects, or in freshly excised tissue with minimum preparation and processing, are important for future diagnosis and surgical guidance in the clinical setting, particularly for application to cancer. Whilst microscopy methods continue to advance on the cellular scale and medical imaging is well established on the scale of the whole tumor or organ, it is attractive to consider imaging the tumor environment on the micro-scale, between that of cells and whole tissues. Such a scenario is ideally suited to optical coherence tomography (OCT), with the twin attractions of requiring little or no tissue preparation, and in vivo capability. OCT’s intrinsic scattering contrast reveals many morphological features of tumors, but is frequently ineffective in revealing other important aspects, such as microvasculature, or in reliably distinguishing tumor from uninvolved stroma. To address these shortcomings, we are developing several advances on the basic OCT approach. We are exploring speckle fluctuations to image tissue microvasculature and we have been developing several parametric approaches to tissue micro-scale characterization. Our approaches extract, from a three-dimensional OCT data set, a two-dimensional image of an optical parameter, such as attenuation or birefringence, or a mechanical parameter, such as stiffness, that aids in characterizing the tissue. This latter method, termed optical coherence elastography, parallels developments in ultrasound and magnetic resonance imaging. Parametric imaging of birefringence and of stiffness both show promise in addressing the important issue of differentiating cancer from uninvolved stroma in breast tissue.

When used for intra-operative imaging of residual basal cell carcinomas (BCCs), reflectance confocal microscopy (RCM) is limited to detection of relatively large tumors. Small tumors remain hidden in the surrounding bright dermis. Fluorescence confocal microscopy (FCM) may improve the sensitivity for detecting small tumors. Fluorescein enhances cell cytoplasm contrast in fluorescence confocal images, but has had limited clinical impact on imaging BCCs in vivo because there is a lack of a well-defined protocol (concentration and application time) that can be effectively used for intraoperative imaging. We conducted an ex vivo study, using discarded tissue from Mohs surgery and a benchtop FCM with 488nm wavelength for excitation and 521nm detection for imaging Concentrations of 6, 0.6 and 0.6 mM with immersion times of 5, 15, 30, and 60 seconds were repeatedly tested (total of 76 specimens).. The 0.6 mM and immersion time of 60 seconds showed that cellular cytoplasm can be labeled with controlled saturation and without leaving the yellow color on the surface of the tissue. Initial results show that, fluorescein may enhance cellular structures contrast relative to other normal dermal structures, improving the detection of small BCCs. This study provide an optimized set of parameters for subsequently testing of topical application in vivo for intraopertive imaging of BCCs.

The use of multiphoton interactions in biological tissue for imaging contrast requires highly sensitive optical measurements. These often involve signal processing and filtering steps between the photodetector and the data acquisition device, such as photon counting and lock-in amplification. These steps can be implemented as real-time digital signal processing (DSP) elements on field-programmable gate array (FPGA) devices, an approach that affords much greater flexibility than commercial photon counting or lock-in devices. We will present progress toward developing two new FPGA-based DSP devices for multiphoton and time-resolved microscopy applications. The first is a high-speed multiharmonic lock-in amplifier for transient absorption microscopy, which is being developed for real-time analysis of the intensity-dependence of melanin, with applications in vivo and ex vivo (noninvasive histopathology of melanoma and pigmented lesions). The second device is a kHz lock-in amplifier running on a low cost ($50--$200) development platform. It is our hope that these FPGA-based DSP devices will enable new, high-speed, low-cost applications in multiphoton and time-resolved microscopy.

Confocal mosaicing microscopy (CMM) enables rapid imaging of large areas of fresh tissue ex vivo without the processing that is necessary for conventional histology. When performed with fluorescence mode using acridine orange (nuclear specific dye) it enhances nuclei-to-dermis contrast that enables detection of all types of BCCs including thin strands of infiltrative basal cell carcinomas (BCCs). Thus far, this technique has been mostly validated in research setting for the analysis of BCC tumor margins. Recently, CMM has been adopted and implemented in real clinical settings by some surgeons as an alternative tool to frozen section (FS) during Mohs surgery. In this review article we summarize the development of CMM guided imaging of ex vivo tissues from bench to bedside. We also present its current state of application in routine clinical workflow not only for the assessment of BCC margin but also for other skin cancers such as melanoma, SCC, and some infectious diseases where FS is not routinely performed. Lastly, we also discuss the potential limitations of this technology as well as future developments. As this technology advances further, it may serve as an adjunct to standard histology and enable rapid surgical pathology of skin cancers at the bedside.

In this report, optical biopsy was applied to diagnose human brain cancer in vitro for the identification of brain cancer from normal tissues by native fluorescence and Stokes shift spectra (SSS). 77 brain specimens including three types of human brain tissues (normal, glioma and brain metastasis of lung cancers) were studied. In order to observe spectral changes of fluorophores via fluorescence, the selected excitation wavelength of UV at 300 and 340 nm for emission spectra and a different Stokes Shift spectra with intervals Δλ = 40 nm were measured. The fluorescence spectra and SSS from multiple key native molecular markers, such as tryptophan, collagen, NADH, alanine, ceroid and lipofuscin were observed in normal and diseased brain tissues. Two diagnostic criteria were established based on the ratios of the peak intensities and peak position in both fluorescence and SSS spectra. It was observed that the ratio of the spectral peak intensity of tryptophan (340 nm) to NADH (440 nm) increased in glioma, meningioma (benign), malignant meninges tumor, and brain metastasis of lung cancer tissues in comparison with normal tissues. The ratio of the SS spectral peak (Δλ = 40 nm) intensities from 292 nm to 366 nm had risen similarly in all grades of tumors.

Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous fluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from imaging of native fluorescence has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Multispectral intrinsic fluorescence imaging was applied to patient olfactory neurosphere-derived cells, cell model of a human metabolic disease MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like syndrome). By using an endogenous source of contrast, subtle metabolic variations have been detected between living cells in their full morphological context which made it possible to distinguish healthy from diseased cells before and after therapy. Cellular maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant cell subpopulations, in particular a subpopulation with compromised mitochondrial function. The versatility of our method is further illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent.

Imaging technologies working in the spatial frequency domain are becoming increasingly popular for generating wide-field optical property maps, enabling further analysis of tissue parameters such as absorption or scattering. While acquisition methods have witnessed a very rapid growth and are now performing in real-time, processing methods are yet slow preventing information to be acquired and displayed in real-time. In this work, we present solutions for rapid inverse problem solving for optical properties by use of advanced look-up tables. In particular, we present methods and results from a dense, linearized look-up table and an analytical representation that currently run 100 times faster than the standard method and within 10% in both absorption and scattering. With the resulting computation time in the tens of milliseconds range, the proposed techniques enable video-rate feedback of real-time techniques such as snapshot of optical properties (SSOP) imaging, making full video-rate guidance in the clinic possible.

In this study, Stokes shift spectroscopy (S3) is used for measuring the aggressiveness of malignant tumors. S3 is an optical tool which utilizes the difference between the emission wavelength (λem) and the absorption wavelength (λabs) (the Stokes shift) to give a fixed wavelength shift (Δλs).Our analysis of tumor samples using S3 shows grade 3 (high grade) cancers consistently have increased relative tryptophan content compared to grade 1 or 2 tumors. This technique may be a useful tool in the evaluation of a patient’s cancer.

Tryptophan is an extremely important amino acid for a variety of biological functions in living organisms. Changes in the concentration of this amino acid can point to identification of cancerous tissues or even confirm symptoms of depression in patients. Therefore it is extremely important to identify and quantify tryptophan concentrations in human blood as well as in in-vivo diagnostic studies. Here a reflection based terahertz pulsed spectroscopy system was used to study the interaction of THz pulses with cancerous cells to gauge the possibility of using L-tryptophan as a biomarker for THz sensing of diseases. Initial measurements were performed on human colon adenocarcinoma cells and human breast cancer cells cultivated on glass slides. The glass slides utilized in the growth process limited the measurements not only to reflection based geometries but also limited the analysis of the samples in the frequency domain due to the highly absorbing nature of glass in the THz region. The useful bandwidth was limited to frequencies below 0.6THz which prohibited us from investigating the effects of L-tryptophan in these samples. Even with the limited frequency range the measurements show that there are slight differences in the transmission of the THz pulse through different samples.

The purpose of this study is to evaluate the effect of a high-lipid diet on elasticity of adipose tissue. We employed dual Raman/Brillouin microspectroscopy to analyze brown and white adipose tissues obtained from adult rats. The rats were divided into two groups, one of which received a high-fat feed, while the other served as a control. We hypothesized that the changes in the elasticity of adipose tissues between the two groups can be successfully assessed using Brillouin spectroscopy. We found that the brown adipose tissue possessed a lesser Brillouin shift than the white adipose within each group and that the elastic modulus of both adipose tissues increases in the high-fat diet group. The Raman spectra provided supplementary chemical information and indicated an increase in the lipid-to-protein ratio in the brown adipose, but not in the white adipose.

We explored the depth penetration in tissue-mimicking intralipid-based phantoms in SWIR (800-1650 nm) using a hyperspectral imaging system composed from a 2D CCD camera coupled to a microscope. Hyperspectral images in transmission and reflection geometries were collected with a spectral resolution of 5.27 nm and a total acquisition time of 3 minutes or less that minimized artifacts from sample drying. Michelson spatial contrast was used as a metric to evaluate light penetration. Results from both transmission and reflection geometries consistently revealed the highest spatial contrast in the wavelength range of 1300 to 1350 nm.

Inducing angiogenesis is one hallmark of cancer. Tumor induced neovasculature is often characterized as leaky, tortuous and chaotic, unlike a highly organized normal vasculature. Additionally, in the course of carcinogenesis, angiogenesis precedes a visible lesion. Tumor cannot grow beyond 1-2 mm in diameter without inducing angiogenesis. Therefore, capturing the event of angiogenesis may aid early detection of pre-cancer –important for better treatment prognoses in regions that lack the resources to manage invasive cancer. In this study, we imaged the neovascularization in vivo in a spontaneous hamster cheek pouch carcinogen model using a, non-invasive, label-free, high resolution, reflected-light spectral darkfield microscope. Hamsters’ cheek pouches were painted with 7,12-Dimethylbenz[a]anthracene (DMBA) to induce pre-cancerous to cancerous changes, or mineral oil as control. High resolution spectral darkfield images were obtained over the course of pre-cancer development and in control cheek pouches. The vasculature was segmented with a multi-scale Gabor filter with an 85% accuracy compared with manually traced masks. Highly tortuous vasculature was observed only in the DMBA treated cheek pouches as early as 6 weeks of treatment. In addition, the highly tortuous vessels could be identified before a visible lesion occurred later during the treatment. The vessel patterns as determined by the tortuosity index were significantly different from that of the control cheek pouch. This preliminary study suggests that high-resolution darkfield microscopy is promising tool for pre-cancer and early cancer detection in low resource settings.

Particle size analyzers based on laser scattering commonly make use of light diffraction and scattering around the particle considered in its medium. For particle size below 50 μm, Fraunhofer theory must be abandoned in favor of Mie model, which requires to know the complex refractive index of both the particle and the medium. In this paper, we demonstrate that particle size characterization can be realized by measuring the macroscopic Raman spectral response of the whole set of particles excited by a laser beam. We use a home-made setup based on coherent anti-Stokes Raman scattering (CARS) and having a 0.36 cm^-1 spectral resolution, in which the laser source is a dual-output infrared nanosecond supercontinuum source (1064 nm monochromatic pump wave, 1100-1640 nm broadband Stokes wave). The samples are latex beads in water with different diameters (20 nm, 50 nm, 100 nm, 5 μm). The C-H stretching line around 3050 cm^-1 is studied. For this vibration, we study the variation of both the CARS central frequency and linewidth as a function of the particles size. A quasi linear increase of the linewidth with the inverse of the diameter is measured. A difference of 15 cm^-1 is obtained between beads with diameters of 5 μm and 20 nm respectively. The physical phenomena at the origin of this difference are discussed, especially considering the contributions of the center and of the boundaries of the object to the global Raman response.

Optical spectroscopy and hyperspectral imaging have shown the theoretical potential to discriminate between cancerous and non-cancerous tissue with high sensitivity and specificity. To date, these techniques have not been able to be effectively translated to endoscope platforms. Hyperspectral imaging of the fluorescence excitation spectrum represents a new technology that may be well-suited for endoscopic implementation. However, the feasibility of detecting differences between normal and cancerous mucosa using fluorescence excitation-scanning hyperspectral imaging has not been evaluated. The objective of this pilot study was to evaluate the changes in the fluorescence excitation spectrum of resected specimen pairs of colorectal adenocarcinoma and normal colorectal mucosa. Patients being treated for colorectal adenocarcinoma were enrolled. Representative adenocarcinoma and normal colonic mucosa specimens were collected from each case. Specimens were flash frozen in liquid nitrogen. Adenocarcinoma was confirmed by histologic evaluation of H&E permanent sections. Hyperspectral image data of the fluorescence excitation of adenocarcinoma and surrounding normal tissue were acquired using a custom microscope configuration previously developed in our lab. Results demonstrated consistent spectral differences between normal and cancerous tissues over the fluorescence excitation spectral range of 390-450 nm. We conclude that fluorescence excitation-scanning hyperspectral imaging may offer an alternative approach for differentiating adenocarcinoma and surrounding normal mucosa of the colon. Future work will focus on expanding the number of specimen pairs analyzed and will utilize fresh tissues where possible, as flash freezing and reconstituting tissues may have altered the autofluorescence properties.

Both Optical Coherence Tomography (OCT) and Single Fiber Reflectance Spectroscopy (SFR) are used to determine various optical properties of tissue. We developed a method combining these two techniques to measure the scattering anisotropy (g1) and γ (=1-g2/1-g1), related to the 1st and 2nd order moments of the phase function. The phase function is intimately associated with the cellular organization and ultrastructure of tissue, physical parameters that may change during disease onset and progression. Quantification of these parameters may therefore allow for improved non-invasive, in vivo discrimination between healthy and diseased tissue. With SFR the reduced scattering coefficient and γ can be extracted from the reflectance spectrum (Kanick et al., Biomedical Optics Express 2(6), 2011). With OCT the scattering coefficient can be extracted from the signal as a function of depth (Faber et al., Optics Express 12(19), 2004). Consequently, by combining SFR and OCT measurements at the same wavelengths, the scattering anisotropy (g) can be resolved using µs’= µs*(1-g). We performed measurements on a suspension of silica spheres as a proof of principle. The SFR model for the reflectance as a function of the reduced scattering coefficient and γ is based on semi-empirical modelling. These models feature Monte-Carlo (MC) based model constants. The validity of these constants - and thus the accuracy of the estimated parameters - depends on the phase function employed in the MC simulations. Since the phase function is not known when measuring in tissue, we will investigate the influence of assuming an incorrect phase function on the accuracy of the derived parameters.

We present an ultra-simple miniature fiber optic probe to measure spatially and spectrally resolved diffuse reflectance in the sub-diffuse regime (i.e. measurements with source-detector separation less than a transport mean free path) in-vivo. This probe has a robust and simple design with a small footprint (<.5 mm diameter). We show that our probe has sensitivity to structures scattering light an order of magnitude smaller than the diffraction limit, and thus can be used to quantify alterations in the very smallest structures in tissue (e.g. organelles, chromatin, collagen fibers, etc.). Specifically, the probe samples the spatial profile of diffuse reflectance in the sub-diffusion regime (P(r), r<<1 mm). P(r) can be used to quantify the entire shape of the phase function, F(θ). The shape of the refractive index correlation function Bn(rd) (through which the spatial distribution of mass is defined) can be analytically derived from the shape of F(θ) through application of the Born approximation. Therefor measurements of P(r) can elucidate F(θ) and Bn(rd). This ability has tremendous potential for use as a diagnostic tool and broad applications for probing the nanoscale environment of tissue in-vivo.

Mueller matrix imaging along with polar decomposition method was employed for the colonic cancer detection by polarized light in the near-infrared spectral range (700–1100 nm). A high-speed (<5s) Muller matrix imaging system with dual-rotating waveplates was developed. 16 (4 by 4) full Mueller matrices of the colonic tissues (i.e., normal and caner) were acquired. Polar decomposition was further implemented on the 16 images to derive the diattentuation, depolarization, and the retardance images. The decomposed images showed clear margin between the normal and cancerous colon tissue samples. The work shows the potential of near-infrared Mueller matrix imaging for the early diagnosis and detection of malignant lesions in the colon.

Polarization gating is a popular and widely used technique in biomedical optics to sense superficial tissues (collinear detection), deeper volumes (cross-linear detection), and also selectively probe deeper volumes (using elliptically polarized light). As opposed to the conventional linearly polarized illumination, we propose a new protocol of polarization gating that combines co-elliptical and counter-elliptical measurements to selectively enhance contrast of the images. In vivo experiments were performed on skin abnormalities of volunteers (to selectively probe and access subsurface information).

Polarization imaging techniques are recognized as potentially powerful tools to detect the structural changes of biological tissues. Meanwhile, spectral features of the scattered light can also provide abundant microstructural information, therefore can be applied in biomedical studies. In this paper, we adopt the polarization reflectance spectral imaging to analyze the microstructural changes of hydrolyzing skeletal muscle tissues. We measure the Mueller matrix, which is a comprehensive description of the polarization properties, of the bovine skeletal muscle samples in different periods of time, and analyze its behavior using the multispectral Mueller matrix transformation (MMT) technique. The experimental results show that for bovine skeletal muscle tissues, the backscattered spectral MMT parameters have different values and variation features at different stages. We can also find the experimental results indicate that the stages of hydrolysis for bovine skeletal muscle samples can be judged by the spectral MMT parameters. The results presented in this work show that combining with the spectral technique, the MMT parameters have the potential to be used as tools for meat quality detection and monitoring.

Degradation and destruction of articular cartilage is the etiology of osteoarthritis (OA), an entity second only to cardiovascular disease as a cause of disability in the United States. Joint mechanics and cartilage biochemistry are believed to play a role in OA; an optical tool to detect structural and chemical changes in articular cartilage might offer benefit for its early detection and treatment. The objective of the present study was to identify the spectral changes in intrinsic ultraviolet (UV) fluorescence of cartilage that occur after proteolytic digestion of cartilage. Bovine articular cartilage samples were incubated in varying concentrations of collagenase ranging from 10ug/mL up to 5mg/mL for 18 hours at 37°C, a model of OA. Pre- and post-incubation measurements were taken of the UV excitation-emission spectrum of each cartilage sample. Mechanical tests were performed to determine the pre- and post-digestion force/displacement ratio associated with indentation of each sample. Spectral changes in intrinsic cartilage fluorescence and stiffness of the cartilage were associated with proteolytic digestion. In particular, changes in the relative intensity of fluorescence peaks associated with pentosidine crosslinks (330 nm excitation, 390 nm emission) and tryptophan (290 nm excitation, 340 nm emission) were found to correlate with different degrees of cartilage digestion and cartilage stiffness. In principle, it may be possible to use UV fluorescence spectral data for early detection of damage to articular cartilage, and as a surrogate measure for cartilage stiffness.

Lower extremity ulcers are one of the most common complications that not only affect many people around the world but also have huge impact on economy since a large amount of resources are spent for treatment and prevention of the diseases. Clinical studies have shown that reduction in the wound size of 40% within 4 weeks is an acceptable progress in the healing process. Quantification of the wound size plays a crucial role in assessing the extent of healing and determining the treatment process. To date, wound healing is visually inspected and the wound size is measured from surface images. The extent of wound healing internally may vary from the surface. A near-infrared (NIR) optical imaging approach has been developed for non-contact imaging of wounds internally and differentiating healing from non-healing wounds. Herein, quantitative wound size measurements from NIR and white light images are estimated using a graph cuts and region growing image segmentation algorithms. The extent of the wound healing from NIR imaging of lower extremity ulcers in diabetic subjects are quantified and compared across NIR and white light images. NIR imaging and wound size measurements can play a significant role in potentially predicting the extent of internal healing, thus allowing better treatment plans when implemented for periodic imaging in future.

In recent conflicts, battlefield injuries consist largely of extensive soft injuries from blasts and high energy projectiles, including gunshot wounds. Repair of these large, traumatic wounds requires aggressive surgical treatment, including multiple surgical debridements to remove devitalised tissue and to reduce bacterial load. Identifying those patients with wound complications, such as infection and impaired healing, could greatly assist health care teams in providing the most appropriate and personalised care for combat casualties.

Candidate technologies to enable this benefit include the fusion of imaging and optical spectroscopy to enable rapid identification of key markers. Hence, a novel system based on IR negative contrast imaging (NCI) is presented that employs an optical parametric oscillator (OPO) source comprising a periodically-poled LiNbO₃ (PPLN) crystal. The crystal operates in the shortwave and midwave IR spectral regions (ca. 1.5 – 1.9 μm and 2.4 – 3.8 μm, respectively). Wavelength tuning is achieved by translating the crystal within the pump beam. System size and complexity are minimised by the use of single element detectors and the intracavity OPO design. Images are composed by raster scanning the monochromatic beam over the scene of interest; the reflection and/or absorption of the incident radiation by target materials and their surrounding environment provide a method for spatial location. Initial results using the NCI system to characterise wound biopsies are presented here.

Many research works based on fluorescence spectroscopy have proven its potential in the diagnosis of various diseases using the spectral signatures of the native key fluorophores such as tryptophan, tyrosine, collagen, NADH, FAD and porphyrin. These fluorophores distribution, concentration and their conformation may be changed depending upon the pathological and metabolic conditions of cells and tissues. In this study, we have made an attempt to characterize the blood plasma of normal subject and oral cancer patients by native fluorescence spectroscopy at 280 nm excitation. Further, the fluorescence data were analyzed by employing the multivariate statistical method - linear discriminant analyses (LDA) using leaves one out cross validation method. The results illustrate the potential of fluorescence spectroscopy technique in the diagnosis of oral cancer using blood plasma.

Urine is considered diagnostically important for tits native fluorophores and they vary in their distribution, concentration and physiochemical properties, depending upon the metabolic condition of the subject. In this study, we have made an attempt, to characterize the urine of normal subject and diabetic patients under medication by native fluorescence spectroscopy at 280 nm excitation. Further, the fluorescence data were analyzed employing the multivariate statistical method linear discriminant analysis (LDA) using leave one out cross validation method. The results were promising in discriminating diabetic urine from that of normal urine. This study in future may be extended to check the feasibility in ruling out the coexisting disorders such as cancer.

Colorectal cancer is the United States 3rd leading cancer in death rates.1 The current screening for colorectal cancer is an endoscopic procedure using white light endoscopy (WLE). There are multiple new methods testing to replace WLE, for example narrow band imaging and autofluorescence imaging.2 However, these methods do not meet the need for a higher specificity or sensitivity. The goal for this project is to modify the presently used endoscope light source to house 16 narrow wavelength LEDs for spectral imaging in real time while increasing sensitivity and specificity.
The process to do such was to take an Olympus CLK-4 light source, replace the light and electronics with 16 LEDs and new circuitry. This allows control of the power and intensity of the LEDs. This required a larger enclosure to house a bracket system for the solid light guide (lightpipe), three new circuit boards, a power source and National Instruments hardware/software for computer control.
The results were a successfully designed retrofit with all the new features. The LED testing resulted in the ability to control each wavelength’s intensity. The measured intensity over the voltage range will provide the information needed to couple the camera for imaging.
Overall the project was successful; the modifications to the light source added the controllable LEDs. This brings the research one step closer to the main goal of spectral imaging for early detection of colorectal cancer. Future goals will be to connect the camera and test the imaging process.

The supercontinuum laser light source has many advantages over other light sources, including broad spectral range. Transmission images of paired normal and malignant breast tissue samples from two patients were obtained using a Leukos supercontinuum (SC) laser light source with wavelengths in the second and third NIR optical windows and an IR- CCD InGaAs camera detector (Goodrich Sensors Inc. high response camera SU320KTSW-1.7RT with spectral response between 900 nm and 1,700 nm). Optical attenuation measurements at the four NIR optical windows were obtained from the samples.

The paper focus on the various algorithms on to unravel the fluorescence spectra by unmixing methods to identify cancerous and normal human tissues from the measured fluorescence spectroscopy. The biochemical or morphologic changes that cause fluorescence spectra variations would appear earlier than the histological approach; therefore, fluorescence spectroscopy holds a great promise as clinical tool for diagnosing early stage of carcinomas and other deceases for in vivo use. The method can further identify tissue biomarkers by decomposing the spectral contributions of different fluorescent molecules of interest. In this work, we investigate the performance of blind source un-mixing methods (backward model) and spectral fitting approaches (forward model) in decomposing the contributions of key fluorescent molecules from the tissue mixture background when certain selected excitation wavelength is applied. Pairs of adenocarcinoma as well as normal tissues confirmed by pathologist were excited by selective wavelength of 340 nm. The emission spectra of resected fresh tissue were used to evaluate the relative changes of collagen, reduced nicotinamide adenine dinucleotide (NADH), and Flavin by various spectral un-mixing methods. Two categories of algorithms: forward methods and Blind Source Separation [such as Principal Component Analysis (PCA) and Independent Component Analysis (ICA), and Nonnegative Matrix Factorization (NMF)] will be introduced and evaluated. The purpose of the spectral analysis is to discard the redundant information which conceals the difference between these two types of tissues, but keep their diagnostically significance. The facts predicted by different methods were compared to the gold standard of histopathology. The results indicate that these key fluorophores within tissue, e.g. tryptophan, collagen, and NADH, and flavin, show differences of relative contents of fluorophores among different types of human cancer and normal tissues. The sensitivity, specificity, and receiver operating characteristic (ROC) are finally employed as the criteria to evaluate the efficacy of these methods in cancer detection. The underlying physical and biological basis for these optical approaches will be discussed with examples. This ex vivo preliminary trial demonstrates that these different criteria from different methods can distinguish carcinoma from normal tissues with good sensitivity and specificity while among them, we found that ICA appears to be the superior method in predication accuracy.

Cervical cancer is considered as the second most commonly occurring malignancy among women, next to breast cancer. It is well known that most of the cancer patients diagnosed with advanced stages and there is a pressing need for improved methods to detect cancer at its initial stages. Many techniques have been adopted for the diagnosis of cervical cancer. Among these, fluorescence polarization spectroscopy is a complementary technique of fluorescence spectroscopy which helps us to elucidate the spectral characteristics which highly depend on pH, viscosity and local environment. Since urine has many metabolites and the measurement of native fluorescence of urine, in principle, able to provide an indication of a number of health conditions, attempts were made to study fluorescence anisotropic characterization of the human urine of cervical cancer patients and normal subjects. Significant differences were observed between the anisotropic and polarization values of cancer subjects and normal subjects.

Stokes shift spectroscopy has been considered as a potential tool in characterization of multiple components present in tissues and biofluids. Since, the intensity and resolution of the fluorophores depends on the Stokes shift, different opinion has been reflected by the researchers in fixing the Stokes shift. Also, not many studies have been reported on the characterization of biofluids and especially on the diagnosis of cancer. Urine is considered as an important diagnostic biofluid as it is rich in many metabolites where many of them are native fluorophores. In this study, we aimed at characterizing the urine of normal subjects and patients with cervical cancer as function of different Stokes shift. It is observed that Neopterin and Riboflavin are the main fluorophores contribute to the variation between normal and cervical cancer subjects. Ratio variables based linear discriminant analysis shows that the Stokes shift of 40 nm and 60 nm may be considered for better characterization with better signal to noise ratio when compared to others.

Raman spectroscopy has become widely used for diagnostic purpose of breast, lung and brain cancers. This report introduced a new approach based on spatial frequency spectra analysis of the underlying tissue structure at different stages of brain tumor. Combined spatial frequency spectroscopy (SFS), Resonance Raman (RR) spectroscopic method is used to discriminate human brain metastasis of lung cancer from normal tissues for the first time. A total number of thirty-one label-free micrographic images of normal and metastatic brain cancer tissues obtained from a confocal micro- Raman spectroscopic system synchronously with examined RR spectra of the corresponding samples were collected from the identical site of tissue. The difference of the randomness of tissue structures between the micrograph images of metastatic brain tumor tissues and normal tissues can be recognized by analyzing spatial frequency. By fitting the distribution of the spatial frequency spectra of human brain tissues as a Gaussian function, the standard deviation, σ, can be obtained, which was used to generate a criterion to differentiate human brain cancerous tissues from the normal ones using Support Vector Machine (SVM) classifier. This SFS-SVM analysis on micrograph images presents good results with sensitivity (85%), specificity (75%) in comparison with gold standard reports of pathology and immunology. The dual-modal advantages of SFS combined with RR spectroscopy method may open a new way in the neuropathology applications.

Resonance Raman (RR) spectroscopic technique has a high potential for label-free and in-situ detection of biomedical lesions in vivo. This study evaluates the ability of RR spectroscopy method as an optical histopathology tool to detect the atherosclerotic plaque states of abdominal aorta in vitro. This part demonstrates the RR spectral molecular fingerprint features from different sites of the atherosclerotic abdominal aortic wall tissues. Total 57 sites of five pieces aortic samples in intimal and adventitial wall from an autopsy specimen were examined using confocal micro Raman system of WITec 300R with excitation wavelength of 532nm. The preliminary RR spectral biomarkers of molecular fingerprints indicated that typical calcified atherosclerotic plaque (RR peak at 964cm^-1) tissue; fibrolipid plaque (RR peaks at 1007, 1161, 1517 and 2888cm^-1) tissue, lipid pool with the fatty precipitation cholesterol) with collagen type I (RR peaks at 864, 1452, 1658, 2888 and 2948cm^-1) in the soft tissue were observed and investigated.

ion for various disease diagnosis including cancers. Oral cancer is one of the most common cancers in India and it accounts for one third of the global oral cancer burden. Raman spectroscopy of tissues has gained much attention in the diagnostic oncology, as it provides unique spectral signature corresponding to metabolic alterations under different pathological conditions and micro-environment. Based on these, several studies have been reported on the use of Raman spectroscopy in the discrimination of diseased conditions from their normal counterpart at cellular and tissue level but only limited studies were available on bio-fluids. Recently, optical characterization of bio-fluids has also geared up for biomarker identification in the disease diagnosis. In this context, an attempt was made to study the metabolic variations in the blood, urine and saliva of oral cancer patients and normal subjects using Raman spectroscopy. Principal Component based Linear Discriminant Analysis (PC-LDA) followed by Leave-One-Out Cross-Validation (LOOCV) was employed to find the statistical significance of the present technique in discriminating the malignant conditions from normal subjects.

Actinic cheilitis is a potentially malignant disorder that mostly affects the vermilion border of the lower lip and can lead to squamous cell carcinoma. Because of its heterogeneous clinical aspect, it is difficult to indicate representative biopsy area. Late diagnosis is a limiting factor of therapeutic possibilities available to treat oral cancer. The diagnosis of actinic cheilitis is mainly based on clinical and histopathological analysis and it is a time consuming procedure to get the results. Information about the organization and chemical composition of the tissues can be obtained using fluorescence lifetime spectroscopy techniques without the need for biopsy. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and allow a quick and non-invasive clinical investigation of injuries and to help clinicians with the early diagnosis of actinic cheilitis. This study aims to evaluate the fluorescence lifetime parameters at the discrimination of three degrees of epithelial dysplasia, the most important predictor of malignant development, described in up to 100% of actinic cheilitis cases.

Diffuse reflectance spectroscopy has been widely used in diagnostic oncology and characterization of laser irradiated tissue. However, still accurate and simple analytical equation does not exist for estimation of diffuse reflectance from turbid media. In this work, a diffuse reflectance lookup table for a range of tissue optical properties was generated using Monte Carlo simulation. Based on the generated Monte Carlo lookup table, an empirical formula for diffuse reflectance was developed using surface fitting method. The variance between the Monte Carlo lookup table surface and the surface obtained from the proposed empirical formula is less than 1%. The proposed empirical formula may be used for modeling of diffuse reflectance from tissue.

Photoaging is the skin premature aging due to exposure to ultraviolet light, which damage the collagen, elastin and can induce alterations on the skin cells DNA, and, then, it may evolve to precancerous lesions, which are widely investigated by fluorescence spectroscopy and lifetime. The fluorescence spectra and fluorescence lifetime analysis has been presented as a technique of great potential for biological tissue characterization at optical diagnostics. The main targeted fluorophores are NADH (nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), which have free and bound states, each one with different average lifetimes. The average lifetimes for free and bound NADH and FAD change according to tissue metabolic alterations and may contribute to a non-invasive clinical investigation of injuries such as skin lesions. These lesions and the possible areas where they may develop can be interrogated using fluorescence lifetime spectroscopy taking into account the variability of skin phototypes and the changes related to melanin, collagen and elastin, endogenous fluorophores which have emissions that spectrally overlap to the NADH and FAD emission. The objective of this study is to assess the variation on fluorescence lifetimes of normal skin at sun exposed and non-exposed areas and associate this variation to the photoaging process.

The optical microscopy is one of the most powerful tool in the analysis of biological systems. The usual transmitted light microscope uses a white light lamp as source, what sometimes does not bring optimal results, making it necessary to introduce filters to change some illumination properties like the color temperature or the color itself. There is, of course, an intrinsic limitation on the use of filters that is the lack of an analogical control on the illumination properties and a practical limitation that depends on the number of available filters. To address this need, we developed an illumination system based on (Red, Green and Blue) RGB LEDs, were the microscope operator can control the intensity of each one independently and manually. This paper details the developed system and describes the methods used to compare quantitatively the images acquired while using the standard white light illumination and the images obtained with the developed system. To quantify the contrast, we calculated the relative population standard deviation for the intensities of each channel of the RGB image. This procedure allowed us to compare and understand the major advantages of the developed illumination system. All analysis methods have shown that a contrast enhancement can be obtained under the RGB LEDs light. The presented illumination allowed us to visualize the structures in different samples with a better contrast without the need of any additional optical filters.

In our study, the skin canceration processes induced by UVB were analyzed from the perspective of tissue spectrum. A home-made Raman spectral system with a millimeter order excitation laser spot size combined with a multivariate statistical analysis for monitoring the skin changed irradiated by UVB was studied and the discrimination were evaluated. Raman scattering signals of the SCC and normal skin were acquired. Spectral differences in Raman spectra were revealed. Linear discriminant analysis (LDA) based on principal component analysis (PCA) were employed to generate diagnostic algorithms for the classification of skin SCC and normal. The results indicated that Raman spectroscopy combined with PCA-LDA demonstrated good potential for improving the diagnosis of skin cancers.

The supercontinuum laser light source has many advantages over other light sources, including broad spectral range. Transmission images of paired normal and malignant breast tissue samples from two patients were obtained using a Leukos supercontinuum (SC) laser light source with wavelengths in the second and third NIR optical windows and an IR- CCD InGaAs camera detector (Goodrich Sensors Inc. high response camera SU320KTSW-1.7RT with spectral response between 900 nm and 1,700 nm). Optical attenuation measurements at the four NIR optical windows were obtained from the samples.