Application of microarrays for single nucleotide polymorphims (SNPs) has a limited appeal currently due to low reliability of experimental results. Theoretical and experimental studies of surface hybridization of heterozygous samples allow us to identify two factors of observed instabilities. First, reactions may not reach thermodynamic equilibrium in the course of the experiment and second, competitive displacement of low affinity species by high affinity species is the mechanism defining specificity of molecular recognition. Here we describe a real time optical detection arrangement that facilitated the detection of competitive displacement between a wild-type target and a SNP target. Results show that even when the SNP is an order of magnitude lower in concentration (100 pM) then the wild-type target, the kinetics of the SNP hybridization affects hybridization of the wild-type target. Additionally, results show that observed binding kinetics can be altered by adjusting the concentration of the SNP without changing the concentration of the wild-type target. These results have significance when considering what needs to be accounted for when analyzing real time hybridization data.

Nanomedicine focuses on a new approach to diagnostic and therapeutic strategies. Nanomedical systems distinguish between diseased and healthy cells on a single cell level and perform a programmed function when necessary. Current research in nanomedicine investigates the interaction of nanomedical systems with living systems in order to assess the biological effects both in the short and long term. The unique goal of the nanomedical system is to deliver a gene for in situ manufacturing of therapeutic agents for cellular repair. Treating cells on an individual level illustrates a paradigm shift created from nanotechnology.

We have constructed a new LighTouch^TM device having improved measurement and control of applied force and tissue position in noninvasive probing of human volar side fingertip capillary beds in vivo using near infrared Raman spectroscopy. These improvements have translated into improved capacity to observe and classify the behavior of specific spectral features under tissue and pulse modulation. Using this and other measurements on various other samples in vitro have demonstrated the validity of associating specific functions of the observed spectra with medically relevant quantities like hematocrit and bicarbonate ion. In addition we can say more about how much of the observed tissue modulated spectra must be associated with a "modulation defect" resulting from various errors affecting the accuracy and precision in subtraction of static tissue contributions.

We have used fiber optic probes with global illumination/collection (PhAT probe, Kaiser Optical Systems) and ring illumination/disk collection configurations for transcutaneous Raman spectroscopy of bone tissue. Both illumination/collection schemes can be used for recovery of spectra of subsurface components. In this paper the global illumination configuration provides minimum local power density and so minimizes the probability of damage to specimens, animals or human subjects. It also allows non-destructive subsurface mapping under certain conditions. The ring/disk probe utilizes a ring of laser light and collects Raman scatter from within the diameter of the ring. This design distributes the laser power for efficient heat dissipation and provides a better collection ratio of subsurface to surface components than the global illumination design. For non-invasive tissue spectroscopy the ring/disk design also provides better rejection of fluorescence from melanocytes. We have tested the performance of these Raman probes on polymer model systems and chicken tibiae.

A time-resolved confocal fluorescence spectroscopy system was instrumented utilizing the multi-channel time-correlated single photon counting (TCSPC) technique. The system provided a unique approach to investigate the relationship between wavelength- and time-resolved cell autofluorescence and cellular metabolic status. The experiments were carried out on monolayered cell cultures including normal and cancer ectocervical cells. With UV excitation at 365 nm, the decay of cellular fluorescence can be well described by a dual-exponential function, consisting of a short lifetime component ((tau)₁~ 0.40 - 0.47 ns) and a long lifetime component (((tau)₂ ~ 3.3 - 4.0 ns). By analyzing the decay-associated spectra of the short and long lifetime components, we found that the long lifetime component carried the information of protein-bound NADH and short lifetime component was mainly determined by free NADH with certain interference from bound NADH. Moreover, it was found that the ratio of the amplitudes of two lifetime components, dominated by free/bound NADH, was sensitive to cell metabolism. Overall, this study demonstrated that wavelength- and timeresolved autofluorescence can be potentially used as an important contrast mechanism to detect epithelial pre-cancer.

We report the results of a comparative evaluation of the diagnostic capabilities of autofluorescence, diffuse reflectance, and Raman spectroscopic approaches in differentiating the various types of breast tumors from normal breast tissues. Optical spectra (n=293) were acquired ex-vivo from a total of 75 breast tissue samples belonging to six distinct histopathologic categories: invasive ductal carcinoma, lobular carcinoma, ductal carcinoma in-situ, fibroadenoma, other benign tumors, and normal breast tissue. Autofluorescence, diffuse reflectance, and Raman spectra were measured from the same locations of a given tissue sample. A probability based multivariate statistical algorithm capable of direct multiclass classification was developed to analyze the diagnostic content of the optical spectra measured from the same set of breast tissue sites with these different techniques. The algorithm uses the theory of nonlinear Maximum Representation and Discrimination Feature (MRDF) for feature extraction, and the theory of Sparse Multinomial Logistic Regression (SMLR) for classification. The results of discrimination analyses reveal that the performance of Raman spectroscopy is superior to that of all others in classifying the breast tissues into respective histopathologic categories. The best classification accuracy was observed to be ~96%, 86%, 94%, 98%, 85%, and 100% for invasive ductal carcinoma, lobular carcinoma, ductal carcinoma in-situ, fibroadenoma, benign tumors and normal breast tissues, respectively, on the basis of leave-one-out cross validation, with the overall accuracy being ~97%.

Recently, optical spectroscopy has shown considerable promise to be used as a potential clinical tool for human brain tumor detection and therapeutic guidance. Our group showed for the first time the possibility of using combined autofluorescence and diffuse reflectance spectroscopy and established its applicability for human brain tumor demarcation in previous in vitro and in vivo studies. We report in this paper the results of a clinical study designed to further evaluate the efficacy of the approach for demarcation of brain tumors and tumor margins from normal brain tissues in intra-operative clinical setting. Using a portable system, optical spectra were collected from the brain of 110 patients undergoing craniotomy at the Vanderbilt University Medical Center. Spectral measurements were taken from multiple sites of tumor core, tumor margin and normal areas of brain tissues and the resulting spectra were correlated with the corresponding histopathologic diagnosis. Using histology as the gold standard, a probabilistic multi-class diagnostic algorithm was developed to simultaneously distinguish tumor core and tumor margin from normal brain tissue sites using independent training and validation sets of data. An unbiased estimate of the accuracy of the model indicates that combined autofluorescence and diffuse reflectance spectroscopy was able to distinguish tumor core and tumor margin from normal brain tissues with an average predictive accuracy of ~88%.

Breast cancer continues to be one of the most widely diagnosed forms of cancer amongst women and the second leading type of cancer deaths amongst women. The recurrence rate of breast cancer is highly dependent on several factors including the complete removal of the primary tumor and the presence of cancer cells in involved lymph nodes. The metastatic spread and staging of breast cancer is also evaluated through the nodal assessment of the regional lymphatic system. A portable real-time spectral domain optical coherence tomography system is being presented as a clinical diagnostic tool in the intraoperative delineation of tumor margins as well as for real time lymph node assessment. The system employs a super luminescent diode centered at 1310 nm with a bandwidth of 92 nm. Using a spectral domain detection system, the data is acquired at a rate of 5 KHz / axial scan. The sample arm is a galvanometer scanning telecentric probe with an objective lens (f = 60 mm, confocal parameter = 1.5 mm) yielding an axial resolution of 8.3 &mgr;m and a transverse resolution of 35.0 &mgr;m. Images of tumor margins are acquired in the operating room ex vivo on freshly excised human tissue specimen. This data shows the potential of the use of OCT in defining the structural tumor margins in breast cancer. Images taken from ex-vivo samples on the bench system clearly delineate the differences between clusters of tumor cells and nearby adipose cells. In addition, the data shows the potential for OCT as a diagnostic tool in the staging of cancer metastasis through locoregional lymph node assessment.

A new clinical diagnostic instrument for urea breath test (UBT) based non-invasive detection of Helicobacter Pylori is presented here. Its compact and low cost design makes it an economical and commercial alternative for the more expensive Isotope Ratio Mass Spectrometer (IRMS). The instrument is essentially a two channel non-dispersive IR spectrometer that performs high precision ratio measurements of the two carbon isotopomers (¹²CO₂ and ¹³CO₂) present in exhaled breath. A balanced absorption system configuration was designed where the two channel path lengths would roughly be in the ratio of their concentrations. Equilibrium between the transmitted channel intensities was maintained by using a novel feedback servo mechanism to adjust the length of the ¹³C channel cell. Extensive computational simulations were performed to study the effect of various possible interferents and their results were considered in the design of the instrument so as to achieve the desired measurement precision of 1%. Specially designed gas cells and a custom made gas filling rig were also developed. A complete virtual interface for both instrument control and data acquisition was implemented in LABVIEW. Initial tests were used to validate the theory and a basic working device was demonstrated.

Near-infrared (NIR) optical imaging is an emerging noninvasive modality for breast cancer diagnosis. However, the currently available optical imaging systems towards tomography studies are limited either by instrument portability, patient comfort, or flexibility to image any given tissue volume. Herein, a hand-held based optical imaging system is developed such that it can possibly overcome some of the above limitations. The unique features of the hand-held optical probe are: (i) to perform simultaneous multiple point illumination and detection, thus decreasing the total imaging time and improving the overall signal strength; (ii) to adapt to the contour of tissue surface, thus decreasing the leakage of excitation and emission signal at contact surface; and (iii) to obtain trans-illumination measurements apart from reflectance measurements, thus improving the depth information. The increased detected signal strength as well as total interrogated tissue volume is demonstrated by simulation studies (i.e. forward model) over a 5×10×10 cc slab phantom. The appropriate number and layout of the source and detection points on the probe head is determined and the hand-held optical probe is developed. A frequency-domain ICCD (intensified charge coupled device) detection system, which allows simultaneous multiple points detection, is developed and coupled to the hand-held probe in order to perform fluorescence-enhanced optical imaging of tissue phantoms. In the future, imaging of homogenous liquid phantoms will be used for the assessment of this hand-held system, followed by extensive imaging studies on different phantoms types under various experimental conditions.

We report on development of minimally invasive system for immediate diagnostics of breast cancer and on the results of its pilot clinical testing. The system designed by BioTelligent Inc is based on analysis of optical diffusion spectra (ODS) measured by a probe inserted into breast tissue during standard punch biopsy. Analysis of scattered spectra aimed to distinction of benign tumors from malignant ones is done by original procedure of data processing. Clinical testing of the created diagnostic system has been performed by classification of spectra collected from 146 patients with previously detected mammary neoplasms. The data of ODS study in each patient have been compared to the results of histology. The proposed technique has to date demonstrated sensitivity of 96%, specificity of 80% and diagnostic accuracy of 88%. These values are expected to improve as the data sets continue to grow and more sophisticated data processing is employed.

Needle-based devices, which are in wide clinical use for needle biopsy procedures, may be augmented by suitable optical techniques for the localization and diagnosis of diseased tissue. Tissue refractive index is one optical contrast mechanism with diagnostic potential. In the case of mammary tissue, for example, recent research indicates that refractive index variations between tissue types may be useful for the identification of cancerous tissue. While many coherence-based forward-sensing devices have been developed to detect scattering changes, none have demonstrated refractive index measurement capabilities. We present a novel needle-based device that is capable of simultaneously measuring refractive index and scattering. Coupled to the sample arm of an optical coherence tomography system, the needle device detects the scattering response and optical pathlength through tissue residing in a fixed-width channel. Near-infrared measurements of tissues and materials with known optical properties using a prototype device will be presented. This work demonstrates the feasibility of integrated in vivo measurement of refractive index and scattering in conjunction with existing clinical needle-based devices.

We report the development of a probability-based multi-class diagnostic algorithm to simultaneously distinguish highgrade dysplasia from low-grade dysplasia, squamous metaplasia as well as normal human cervical tissues using nearinfrared Raman spectra acquired in-vivo from the cervix of patients at the Vanderbilt University Medical Center. Extraction of diagnostic features from the Raman spectra uses the recently formulated theory of nonlinear Maximum Representation and Discrimination Feature (MRDF), and classification into respective tissue categories is based on the theory of Sparse Multinomial Logistic Regression (SMLR), a recent Bayesian machine-learning framework of statistical pattern recognition. The algorithm based on MRDF and SMLR was found to provide very good diagnostic performance with a predictive accuracy of ~90% based on leave-one-out cross validation in classifying the tissue Raman spectra into the four different classes, using histology as the "gold standard". The inherently multi-class nature of the algorithm facilitates a rapid and simultaneous classification of tissue spectra into various tissue categories without the need to train and heuristically combine multiple binary classifiers. Further, the probabilistic framework of the algorithm makes it possible to predict the posterior probability of class membership in discriminating the different tissue types.

In the last few years there has been a growing interest in the use of small animal optical molecular imaging systems to conduct preclinical studies. Most of these imaging systems are based on continuous wave (CW) technology to measure the bioluminescence or fluorescence light intensity from optical probes in small animals. The eXplore Opti^TM is currently the only commercially available imaging system based on time domain (TD) technology. In addition to measuring the light intensity, the TD approach provides extra information to help determine the depth and concentration of optical probes in small animals. Furthermore, the TD approach uniquely allows the fluorescence lifetime of a fluorophore-based optical probe to be measured. Recently, our single wavelength eXplore-Opti^TM system has been upgraded to a multi-wavelength (eXplore Optix^TM-MX) system with the addition of 3 laser wavelengths and corresponding filters. This has enabled us to image a variety of fluorophores for different preclinical applications. Preliminary results evaluating the performance of the eXplore-Opti^TM-MX are presented employing fluorophores with different spectral and lifetime characteristics.

Developing the luminous system in a capsule endoscope, it is difficult to obtain an uniform illumination[1] on the observed object because of several reasons: the light pattern of LED is sensitively depend on the driving current, location and projective angles; the optical path of LED light source is not parallel to the optical axis of the nearby imaging lenses; the strong reflection from the inner surface of the dome may saturate the CMOS sensors; the object plane of the observed intestine is not flat. Those reasons induce the over-blooming and deep-dark contrast in a picture and distort the original image strongly. The purpose of the article is to construct a photometric model to analyze the LED projection light pattern, and, furthermore, design a novel multiple LEDs luminous system for obtaining an uniform-brightness image. Several key parameters resulting as illumination uniformity has been taken under the model consideration and proven by experimental results. Those parameters include LED light pattern accuracy, choosing LED position relative to the imaging optical axis, LED numbers, arrangement, and the inner curvature of the dome. The novel structure improves the uniformity from 41% to 71% and reduces the light energy loss under 2%. The progress will help medical professionals to diagnose diseases and give treatment precisely based on the vivid image.

Proteomics based techniques are rapidly emerging as alternative techniques to conventional histo-pathological methods for detection and diagnosis of cancers. Tumor markers are of considerable importance in the study of various cancers. A study of various changes in the protein profile associated with breast cancer will facilitate a better understanding of the various dynamic changes associated with the disease. In our study we have used High Performance Liquid Chromatography coupled with highly sensitive Laser Induced Fluorescence for recording the protein profiles of breast tissue homogenates. The protein profiles were recorded from pathologically certified normal as well as malignant breast tissue samples. The recorded protein profiles were studied by using Principal Component Analysis. Good discrimination of normal, benign and malignant samples was achieved in this pilot study.

In this work the possibilities of infrared thermography for the study of burned human bones of outstanding interest in archaeology and anthropology are explored. The technique used consisted in the illumination of the sample using an infrared solid state laser beam and the observation and the monitoring of the surface temperature with an infrared camera. The bones analyzed were previously thermally treated in a furnace and boiled in water. It is shown that the effect of the thermal treatments can be observed in the infrared images, from which the dynamics of the cooling process of the sample is obtained. It is shown that the cooling process of the samples could be used to identify the possible burning treatment at which a given material could have undergone previously. As an auxiliary technique X-ray diffraction was used to analyze the crystallization of the material and to look for a correlation with different thermal treatments.

Tissue viability represents the balance between O₂ supply and demand. In our previous paper (Mayevsky et al; Proc.SPIE 6083 : z1-z10, 2006) the HbO₂ was added to the multiparametric tissue spectroscope (Mayevsky et al J.Biomedical Optics 9:1028-1045,2004). This parameter provides relative values of microcirculatory blood oxygenation (MC-HbO₂) evaluated by the 2 wavelength reflectometry principle. The advantage of this approach as compared to pulse oximetry is that the measurement is not dependent of the existence of the pulse of the heart. Also in the MC-HbO₂ the information is collected from small vessels providing O₂ to the mitochondria as compared to the pulse oximeter indicating blood oxygenation by the respiratory and cardiovascular systems. In the present study we compared the level of blood oxygenation measured by the pulse oximeter to that measured by the CritiView in the brain exposed to various systemic and localized perturbations of O₂ supply or demand. We exposed gerbils to anoxia, hypoxia, ischemia and terminal anoxia. In addition we measured mitochondrial NADH (surface fluorometry), tissue reflectance, tissue blood flow (laser Doppler flowmetry) from the same site of MC-HbO₂ measurement. A clear connection was found between the two blood oxygenation parameters only when systemic perturbations were used (anoxia, hypoxia and terminal anoxia). Under local events (ischemia) the MC-HbO₂ was responsive while the systemic oxygenation was unchanged. We concluded that MC-HbO₂ has a significant value in interpretation of tissue energy metabolism under pathophysiological conditions.

An optical sensor for the continuous detection of pH in the interstitial fluid was developed. The pH sensing layer is immobilised on the internal wall of a glass capillary which is in series with the microdialysis catheter. Phenol red is the pH indicator and is covalently bound directly to the glass surface by means of the Mannich reaction. An optoelectronic unit, which makes use of a light emitting diode at 590 nm as source and a photodiode as detector, is used for the interrogation of the glass capillary. Optical fibres (core diameter: 200 &mgr;m) are used to couple the sensing capillary with the unit. Effect of the ionic strength was studied and the performance of the sensor in contact with dialysed blood was carefully investigated. Particular attention was given also to the pH recovery rate, which is given by the ratio between the hydrogen ion concentration in the perfusate and the hydrogen ion concentration in the analysed medium. The pH sensor works in the range 6-8 pH units and it is characterised by an accuracy of 0.07 pH units and a response time of the order of the minute. Long term stability was checked and the sensor is perfectly working for a period of three days, when exposed to dialysed blood. In vivo tests on animals and on volunteers are described.

In the emerging fields of high-content and high-throughput single cell analysis for Systems Biology and Cytomics multi- and polychromatic analysis of biological specimens has become increasingly important. Combining different technologies and staining methods polychromatic analysis (i.e. using 8 or more fluorescent colors at a time) can be pushed forward to measure anything stainable in a cell, an approach termed hyperchromatic cytometry. For cytometric cell analysis microscope based Slide Based Cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide. Based on the feature of relocation identical cells can be subsequently reanalyzed. In this manner data on the single cell level after manipulation steps can be collected. In this overview various components for hyperchromatic cytometry are demonstrated for a SBC instrument, the Laser Scanning Cytometer (Compucyte Corp., Cambridge, MA): 1) polychromatic cytometry, 2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), 3) differential photobleaching (differentiating fluorochromes by their different photostability), 4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and 5) photodestruction (destruction of FRET dyes). With the intelligent combination of several of these techniques hyperchromatic cytometry allows to quantify and analyze virtually all components of relevance on the identical cell. The combination of high-throughput and high-content SBC analysis with high-resolution confocal imaging allows clear verification of phenotypically distinct subpopulations of cells with structural information. The information gained per specimen is only limited by the number of available antibodies and by sterical hindrance.

This project comprises the development of a novel polymeric BioMEMS device capable of rapidly detecting FIV in a minimally invasive manner. FIV severely inhibits the infected feline from mounting an immune response, and causes susceptibility to other types of diseases. Vaccines against FIV do exist, but have some strong limitations to their effectiveness; so early detection is the best method to combat the spread of the disease. Current testing methods look for antibodies to the FIV protein p24 in feline blood using established Enzyme Linked ImmunoSorbent Assay (ELISA) protocols. The focus of this research is to design and construct a device that can detect antibodies to p24 in a salivary sample by non-intrusive electrochemical means. The device is constructed upon a silicon substrate with gold microelectrodes coated with polypyrrole (PPy), an electrically conducting and biocompatible polymer. In the current phase of the research, the PPy deposition process has been optimized with regards to film thickness, uniformity and conductivity. Microfluidic channels have been fabricated using SU-8, an epoxy based polymer that enables the test sample and other solutions to pass freely through the device. The PPy will be coated with anti-FIV p24 antibodies that can capture FIV p24 antigens present in a salivary sample. Future research will involve the analysis of PPy/antibody interaction and its effect on functionality. The capture of such antigens will interfere with a reduction-oxidation (redox) reaction in a subsequently added ionic solution. This interference will change the characteristic resistance of the solution yielding a qualitative test for the presence of the viral antigens in the sample and hence determining the occurrence of infection.

Autofluorescence spectroscopy has been a widely explored technique for in vivo and noninvasive diagnosis of pre-cancer lesions in epithelium where 90% cancers originate. For extracting more accurate fluorescence information for cancer diagnosis, depth-resolved fluorescence measurements are crucial to assess NADH and FAD in non-keratinized epithelial layer and collagen in stromal layer, respectively. In this study, we achieved the depth-resolved fluorescence spectral measurements of squamous epithelial tissue based on confocal technique. We found that in non-keratinized epithelial layer the fluorescence signals excited at 405 nm were the combination of NADH and FAD fluorescence and could be used for evaluating the redox ratio. Moreover, we found that confocal time-resolved autofluorescence measurements of epithelial tissue with 405 nm excitations could provide the information on the layered tissue structure. All depth-resolved autofluorescence decays were accurately fitted with a dual-exponential function consisting of a short lifetime (0.4 ~ 0.6 ns) and a long lifetime (3 ~ 4 ns) components. The short lifetime component dominated the decay of non-keratinzied epithelial fluorescence while the decay of the signals from keratinized epithelium and stroma were mainly determined by the long lifetime component. The ratio of the amplitudes of two components could be used to differentiate the layered structure of epithelial tissue. In general, the results in this study demonstrated that the combined depth- and timeresolved fluorescence measurements can produce the information on the layered structure and localized biochemistry of epithelial tissue for the diagnosis of tissue pathology.

A realtime photoacoustic microscopy system consisting of a high-repetition rate pulsed laser, high-frequency (30 MHz) ultrasound array transducer, and realtime receiving system was used to visualize microvessels pulsations over a cardiac cycle. The system offers 100 μm lateral spatial resolution, 25 µm axial spatial resolution, and can image at a rate of 83 frames per second. The system shows promise for visualizing time-varying processes in the microvasculature.

Development of cholesterol biosensors is of great importance in clinical analysis because the concentration of cholesterol in blood is a fundamental parameter for the prevention and diagnosis of a number of clinical disorders such as heart disease, hypertension and arteriosclerosis. In general, determination of cholesterol is based on spectrophotometry; but this method involves complicated procedures and the cost is high because expensive enzyme must be used in each assay. We report here the observation, for the first time, of the enhancement of Europium-Tetracycline complex emission in cholesterol solutions. This enhancement was initially observed with the addition of the enzyme cholesterol oxidase, which produces H₂O₂, the agent driver of the Europium tetracycline complex, to the solution. However, it was found that the enzyme is not needed to enhance the luminescence. A calibration curve was determined, resulting in an easy-handling immobilization method with a cheap stable material. This method shows that the complex can be used as a sensor to determine cholesterol in biological systems with good selectivity, fast response, miniature size, and reproducible results.

Distortion exists in the present capsule endoscope image resulting from the confined space and the wide-angle requirement [8]. Based on the previous two lens works, the optimal design had obtained that the field of view was about 86 degrees , and MTF was about 18% at 100 lp/mm, but distortion would go to -26%. It's difficult to add another lens on the 7mm optical path between the dome and imaging lenses for improving distortion. In order to overcome this problem, we intend to design the optical dome as another optical lens. The original dome is transparent and has an equal thickness, namely without refracting light almost. Our objective in this paper is to design the inner curvature of the dome and associate two aspheric imaging lenses in front of the CMOS sensors to advance the distortion with maintaining field of view and MTF under the same capsule volume. Furthermore, the paper proposes the real object plane of intestine is nearly a curved surface rather than an ideal flat surface. Taking those reasons under consideration, we design three imaging lenses with curved object plane and obtain the field of view is about 86 degrees , MTF is about 26% at 100 lp/mm, and the distortion improve to -7.5%. Adding the dome lens is not only to enhance the image quality, but also to maintain the tiny volume requirement.

Raman spectroscopy was applied to distinguish the spectroscopic information between normal cervical tissues (14) and cervical neoplasia (17), including low grade squamous intraepithelial lesions (6) and high grade squamous intraepithelial lesions (11). Standard pathological sections of these cervical tissues were measured from superficial to stroma layers. We have normalized significant Raman peaks, 1250 and 1579-1656 cm^-1 by taking a ratio over a stationary Raman at 1004 cm^-1, and successfully discriminated between normal and neoplasm cervical tissues.

Raman spectroscopy has been shown to have the potential for providing oxygenated ability of erythrocytes. Raman line at 1638 cm-1 has also been reported as one significant oxygenic indicator for erythrocytes. In this research, we develop the Raman spectroscopic monitoring of the bioeffects of Nitroglycerin on hemoglobin oxygen saturation in a single red blood cell (RBC). Nitroglycerin has been frequently used in the management of angina pectoris. Nitroglycerin liberates nitric oxide (NO) to blood vessels. NO is an oxidizer that easily converts hemoglobin to methemoglobin. The conversion may cause the decrease of oxygenated ability of erythrocytes. In this study, we observed the oxidize state of erythrocytes caused by the over dosage of Nitroglycerin. When the dose of Nitroglycerin exceeds 2x10^-4 M, the oxygenic state of erythrocytes decreases significantly. The Raman spectroscopic results demonstrate the observation of the bioeffects of Nitroglycerin on hemoglobin.

We show that early indicators of osteoarthritis are observed in Raman spectroscopy by probing femur surfaces excised from mouse models of early-onset osteoarthritis. Current clinical methods to examine arthritic joints include radiological examination of the joint, but may not be capable of detecting subtle chemical changes in the bone tissue, which may provide the earliest indications of osteoarthritis. Recent research has indicated that the subchondral bone may have a more significant role in the onset of osteoarthritis than previously realized. We will report the effect of age and defective type II collagen on Raman band area ratios used to describe bone structure and function. The carbonate-to-phosphate ratio is used to assess carbonate substitution into the bone mineral and the mineral-to-matrix ratio is used to measure bone mineralization. Mineral-to-matrix ratios indicate that subchondral bone becomes less mineralized as both the wild-type and Del1 (+/-) transgenic mice age. Moreover, the mineral-to-matrix ratios show that the subchondral bone of Del1 (+/-) transgenic mice is less mineralized than that of the wild-type mice. Carbonate-to-phosphate ratios from Del1 (+/-) transgenic mice follow the same longitudinal trend as wild-type mice. The ratio is slightly higher in the transgenic mice, indicating more carbonate content in the bone mineral. Raman characterization of bone mineralization provides an invaluable insight into the process of cartilage degeneration and the relationship with subchondral bone at the ultrastructural level.

The use of lasers to remotely and non-invasively detect the blood pressure waveform of humans and animals would provide a powerful diagnostic tool. Current blood pressure measurement tools, such as a cuff, are not useful for burn and trauma victims, and animals require catheterization to acquire accurate blood pressure information. The purpose of our sensor method and apparatus invention is to remotely and non-invasively detect the blood pulse waveform of both animals and humans. This device is used to monitor an animal or human's skin in proximity to an artery using radiation from a laser Doppler vibrometer (LDV). This system measures the velocity (or displacement) of the pulsatile motion of the skin, indicative of physiological parameters of the arterial motion in relation to the cardiac cycle. Tests have been conducted that measures surface velocity with an LDV and a signal-processing unit, with enhanced detection obtained with optional hardware including a retro-reflector dot. The blood pulse waveform is obtained by integrating the velocity signal to get surface displacement using standard signal processing techniques. Continuous recording of the blood pulse waveform yields data containing information on cardiac health and can be analyzed to identify important events in the cardiac cycle, such as heart rate, the timing of peak systole, left ventricular ejection time and aortic valve closure. Experimental results are provided that demonstrates the current capabilities of the optical, non-contact sensor for the continuous, non-contact recording of the blood pulse waveform without causing patient distress.

Melanoma is the most aggressive skin cancer and is invariably fatal if left untreated. Melanoma removal at early stages is almost always curative and therefore early detection is essential. Removal of every pigmented lesion is unacceptable for the patient, especially in the case of multiple skin lesions or lesions localized in cosmetically important parts of the body such as the face because of risk of scarring. The development of a technique to detect these changes in a noninvasive way is therefore crucial for melanoma detection. In this study, we have used FT-Raman Spectroscopy to investigate through PCA analysis the alterations in the molecular structure of 90 skin spectra, being 30 Pigmented Nevi, 30 Primary Melanoma, and 30 Metastasis, for 6 patients. For projection of data, the scores (Principal Components) PC1 to PC3 were calculated. PC1 versus PC3 for the 800 to 1800 cm^-1 spectral region. PC1 versus PC2 for the 1200 to 1400 cm^-1 spectral region. In both analysis, we could differentiate the three different types of tissues.

This article presents a novel approach to evaluate two-dimensional histomorphometric studies of biodegradable ceramic particles by means of element-sensitive, three-dimensional and non-destructive synchrotron-microtomography (SCT). An in vivo study was performed in which bone substitute materials (Cerasorb) were implanted in the mandible to support the bone regeneration. After 6 months of implantation samples were prepared and investigated using SCT and subsequent 3D image analysis as well as histological evaluation. A comparison of corresponding tomographical and histological slices delivers information about the newly formed bone and its stage of development. Additionally SCT gives insights into the structural changes of the bony tissue in a given defect and the local biodegradation of the bone substitute material in a three-dimensional manner. This emphasizes the fact that the completion of investigations by 2D histological images with 3D tomographical images is required in order to be able to draw conclusions concerning the influence of different bioceramics on the bone regeneration.

Parallel recording of reflection photoplethysmography (PPG) signals in broad spectral band has been performed, and potential of this approach for assessment of blood microcirculation at various vascular depths is discussed. PPG signals have been simultaneously detected at cw laser wavelengths sets comprising 405 nm, 532 nm, 645 nm, 807 nm and 1064 nm. Different signal baseline responses to breath holding at different wavelengths have been observed, as well as different shapes of the PPG pulses originated from the same heartbeat.

We report an in-vitro autofluorescence spectroscopic study of cow eye tissue to explore the applicability of the approach in discriminating early stage "cancer eye" from normal squamous eye tissues. Significant differences were observed in the autofluorescence signatures between the "cancer eye" and normal eye tissues. The spectral differences were quantified by employing a probability-based diagnostic algorithm developed based on recently formulated theory of Relevance Vector Machine (RVM), a Bayesian machine-learning framework of statistical pattern recognition. The algorithm provided sensitivity and specificity values of 97 ± 2% towards cancer for the training set data based on leave-one-out cross validation and a sensitivity of 97 ± 2% and a specificity of 99 ± 1% towards cancer for the independent validation set data. These results suggest that autofluorescence spectroscopy might prove to be a quantitative in-vivo diagnostic modality for early and accurate diagnosis of "cancer eye" in veterinary clinical setting, which would help improve ranch management from both economic and animal care standpoint.

We report firsthand on innovative developments in non-invasive, biophotonic techniques for a wide range of diagnostic, imaging and treatment options, including the recognition and quantification of cancerous, pre-cancerous cells and chronic inflammatory conditions. These techniques have benefited from the ability to target the affected site by both monochromatic light and broad multiple wavelength spectra. The employment of such wavelength or color-specific properties embraces the fluorescence stimulation of various photosensitizing drugs, and the instigation and detection of identified fluorescence signatures attendant upon laser induced fluorescence (LIF) phenomena as transmitted and propagated by precancerous, cancerous and normal tissue. In terms of tumor imaging and therapeutic and treatment options, we have exploited the abilities of various wavelengths to penetrate to different depths, through different types of tissues, and have explored quantifiable absorption and reflection characteristics upon which diagnostic assumptions can be reliably based and formulated. These biophotonic-based diagnostic, sensing and imaging techniques have also benefited from, and have been further enhanced by, the integrated ability to provide various power levels to be employed at various stages in the procedure. Applications are myriad, including non-invasive, non destructive diagnosis of in vivo cell characteristics and functions; light-based tissue analysis; real-time monitoring and mapping of brain function and of tumor growth; real time monitoring of the surgical completeness of tumor removal during laser-imaged/guided brain resection; diagnostic procedures based on fluorescence life-time monitoring, the monitoring of chronic inflammatory conditions (including rheumatoid arthritis), and continuous blood glucose monitoring in the control of diabetes.

Spatial frequency domain imaging (SFDI), a novel non-contact optical imaging technology, is applied to the detection of bruising on Golden Delicious apples. Quantitative absorption and scattering image maps from 650 to 980 nm are obtained for two levels of bruising severity. While not obviously distinguishable by eye, using SFDI the severity of these bruises, which have been created using a controlled impact device, are distinguishable from each other and from the surrounding non-bruised region. The average scattering and absorption spectra is calculated for the two levels of bruising and compared to the adjacent non-bruised regions. There is a considerable difference in the average reduced scattering coefficients between the bruise and non-bruised regions for the two levels of bruising from 650 to 980 nm.

Nowadays laser sources are largely adopted in dentistry due to their unique properties making them good candidates to substitute traditional scalpel and conventional diamond bur in the surgery of the soft and hard oral tissue, respectively. The large use of laser sources outside the research laboratories without the need of highly specialized personnel can ask for a widespread knowledge of safety issues related to this kind of equipment. The main hazard of accidental exposures regards eyes injury but increasing the power of the laser beam also skin can be involved. Safety legislations in Europe and U.S.A. take into account non ionizing radiations and laser radiation for the hazards for the health deriving from physical agents. Laser safety standards introduce 3 useful parameters for hazard characterization: "Accessible Emission Limit" (AEL), "Maximum Permissible Exposure" (MPE) and "Nominal Ocular Hazard Distance" (NOHD). We measured the MPE and NOHD for Er:YAG and other laser sources currently adopted in dentistry and we compared our results with data elaborated from standards in order to single out safe and comfortable working conditions. In fact an experimental assessment of the hazard parameters and the comparison with those of reference from safety standards turns out to be useful in order to estimate the residual hazard that can be still present after applying all the engineering protection and administrative rules.

With the development of fluorescence spectroscopy, multicenter clinical trials are becoming more common both in the academic and commercial arenas. To ensure the quality of quantitative and device independent results, standardization of the tissue spectra is essential for the comparison of data from various groups. An added concern is the potential degradation of instrumentation during a trial which may affect the instrument's ability to accurately represent the tissue spectra. Our group has recently completed a Phase II clinical trial for the detection of cervical neoplasia using two different generations of spectroscopic devices at multiple sites. Both positive and negative optical standards were used to calibrate the tissue spectra as well as aid in the diagnosis of potential instrumentation problems during the trial. We have also conducted a cross validation study of fiber optic probes, spectroscopic devices, and optical standards for the latest generation of devices. The spectroscopic data of optical standards were analyzed for both the clinical trial and cross validation studies. Results demonstrated perceptible differences in optical standards data between the two generations of spectroscopy devices in the clinical trial, as well as the cross validation study with multiple devices of the same generation. Although the spectra were unexpectedly different, tissue spectra measured with the different systems can be empirically corrected by use of the various optical standards. Device performance during the clinical trial also was a concern; however, with the use of optical calibration standards, instrumentation problems were easily identified. To eliminate the problems associated with instrumentation, we have recently developed real-time quality assurance software to assess the optical calibration standards immediately after acquisition.

Measurement quality assurance plans for optical devices should be a mandatory part of grant funding submissions and should explicitly affect scoring during review. These should include calibration strategy, standards selection strategy, performance verification plan, performance validation plan and thorough preclinical performance validation. A multispectral digital colposcope (MDC) has been designed to collect image data from patients as part of an NIH sponsored clinical trial, based on a technology assessment model. Calibration strategy, standards selection and performance verification methods are presented that may be used as a template for smaller groups or more limited studies. With the MDC, red green and blue fluorescence images are captured under ultraviolet light excitation and red and green images are captured under blue light excitation. Red, green and blue reflectance images are captured under broadband white light illumination from a metal halide lamp in three modes - ordinary reflectance, and with polarized illumination in combination with parallel and cross-polarized filtered imaging. The highly automated system was designed to collect images of the cervix prior to and following the application of acetic acid. Three systems have been built and will be operated in clinics in Vancouver, Canada, Houston, Texas and other locations in the developed and developing world including Nigeria. The system is designed with a comprehensive set of calibration and performance verification standards, based on our experience with large scale multi-center spectroscopy clinical trials and measurements are made frequently prior to and following patient measurements. Automated performance verification procedures are being designed based on measurements made during pilot studies to facilitate larger clinical trials.

Flow cytometry has been instrumental in rapid analysis of single cells since the 1970s. One of the common approaches is the immunofluorescence study involving labeling of cells with antibodies conjugated to organic fluorophores. More recently, as the application of flow cytometry extended from simple cell detection to single-cell proteomic analysis, the need of determining the actual number of antigens in a single cell has driven the flow cytomery technique towards a quantitative methodology. However, organic fluorophores are challenging to use as probes for quantitative detection due to the lack of photostability and of quantitative fluorescence standards. National Institute of Standards and Technologies (NIST) provides a set of fluorescein isothiocyanate (FITC) labeled beads, RM 8640, which is the only nationally recognized fluorescent particle standard. On the other hand, optical characteristics of semiconductor nanocrystals or quantum dots or QDs are superior to traditional dye molecules for the use as tags for biological and chemical fluorescent sensors and detectors. Compelling advantages of QDs include long photostability, broad spectral coverage, easy excitation, and suitability for multiplexed sensing. Recently, novel surface coatings have been developed to render QDs water soluble and bio-conjugation ready, leading to their use as fluorescent tags and sensors for a variety of biological applications including immunolabeling of cells. Here, we describe our approach of using fluorescent semiconductor QDs as a novel tool for quantitative flow cytometry detection. Our strategy involves the development of immuno-labeled QD-conjugated silica beads as "biomimetic cells." In addition to flow cytometry, the QD-conjugated silica beads were characterized by fluorescence microscopy to quantitate the number of QDs attached to a single silica bead. Our approach enables flow cytometry analysis to be highly sensitive, quantitative, and encompass a wide dynamic range of fluorescence detection. Quantitative aspects of the proposed flow cytometery-based approach for measurement of the QD-based biomimetic samples are discussed.

This paper describes the design-for-manufacture (DFM) process for a multi-channel fluorometer product platform. The multi-disciplinary team eliminated the cost of quality by design, using a formal design method, facilitated by Lambda Research Corporation's suite of TraceProTM suite of optical design software. Development of this platform presented rigorous design challenges - from identifying feasible design alternatives to minimizing the exponential cumulative effect of component quality and quantity to optimizing tolerances to thoroughly documenting the design. The design was highly constrained in terms of cost and the ability of the platform to accommodate a breadth of fluorescence-tagged media. Furthermore, the inherently interdisciplinary nature of developing medical devices required a high level of collaboration between scientists and engineers across the areas of optics, mechanics, materials, biology and clinical chemistry. While fluorescence tag technologies enable very sensitive detection of molecules, the anisotropic nature of fluorescence in both intensity and polarization severely complicate system design. TracePro's fluorescence modeling capability enabled adherence to a methodical design process of (1) testing system design alternatives, (2) evaluating off-the- shelf and custom optical component and fluorophore feasibility, and (3) tolerancing for robustness without the cost and time associated with iterative hardware prototyping.

While fundamental research in areas of drug discovery and medical device development will continue to be important, additional emphasis is now being placed on translational research. Here, the scientific discoveries coming from laboratory, clinical, or population studies are being moved into clinical practice. For the most part, this translation is being done with only minimal forethought and planning. This talk will examine models for translational research and the roles and needs of standards in the process. While many of the concepts are valid for all aspects of biomedical research, examples from the optical technology and device area will be highlighted. In particular the NCI Network for Translational Research: Optical Imaging (NTROI) will be mentioned.

We report results of a study demonstrating that swept-source spectral interferometry and spectral phase analysis may be applied to determine quantitatively the concentration of glucose in aqueous solutions. The optical system incorporates a frequency swept laser and fiber-based common-path interferometer and provides a compact, stable experimental system. Spectral phase analysis includes a fast Lomb-Scargle algorithm and a multitaper spectral estimation method that allows for time-frequency analysis. The results show highly sensitive and accurate determination of glucose concentration in solutions with 0.54 mM of resolution and 0.999 correlation coefficient. Swept-source spectral interferometry with spectral phase analysis is a promising methodology for further development of a convenient glucose-sensing method with superior sensitive and accuracy.

For the light source of photocoagulators for ophthalmology, orange laser is more suitable than green laser because of low scattering loss by the crystalline lens, and low absorption by xanthophylls in the retina. We developed two orange fiber lasers (580 nm and 590 nm) to investigate the effect depending on the difference in the range of orange. The 580nm laser is composed of a 1160 nm fiber laser and a Periodically Polled Lithium Niobate (PPLN) crystal for second harmonic generation. The 1160 nm fiber laser beam is focused into the MgO-doped PPLN crystal whose length is 30 mm with 3-pass configuration. Continuous-wave 1.3 W output power of 580 nm was obtained with 5.8 W input power of 1160nm for the first time. The conversion efficiency was 22%. The band width of the second harmonic was 0.006 nm (FWHM). The 590 nm laser is almost the same as 580 nm laser source. In this case we used a Raman shift fiber to generate 1180 nm, and the output power of 590 nm was 1.4 W. We developed an evaluation model of photocoagulator system using these two laser sources. A 700 mW coagulation output power was obtained with this orange fiber laser photocoagulator system. This is enough power for the eye surgery. We have the prospect of the maintenance-free, long-life system that is completely air-cooled. We are planning to evaluate this photocoagulator system in order to investigate the difference between the two wavelengths at the field test.

Time-resolved fluorescence (TRF) measurements from layered biological tissue provide chemical and structural information which may be useful for imaging or single-point tissue diagnostics. While several techniques for analyzing TRF data have been proposed in the literature, a rigorous theoretical evaluation of these approaches has not been performed. In this study we have evaluated the sensitivity and robustness of four methods for analyzing TRF signals: biexponential deconvolution, single exponential deconvolution, Laguerre deconvolution, and direct peak width computation. Each of these analyses was performed on a large dataset of synthetic fluorescence decay curves. Each decay curve was generated by numerically convolving a pre-recorded nitrogen laser pulse with a biexponential decay based on fluorescence lifetimes of colonic mucosa. The relative contribution of each mucosal layer to the total TRF signal as well as the superficial layer's inherent lifetime were varied so as to investigate sensitivity to morphological and biochemical changes representative of a neoplastic disease process. To evaluate robustness, pre-set levels of Gaussian-distributed noise were added to the convolved curves to achieve variations in the signal-to-noise ratio. The relative merits and pitfalls of each analytical method are discussed.

We have fabricated a combined measurement system capable of confocal microscopy and fluorescence spectroscopy to simultaneously evaluate multiple optical characteristics of single fluorescent nanocrystals. The single particle detection sensitivity is demonstrated by simultaneously measuring the dynamic excitation-time-dependent fluorescence intermittency and the emission spectrum of single cadmium selenide/zinc sulfide (CdSe/ZnS) nanocrystals (quantum dots, QDs). Using this system, we are currently investigating the optical characteristics of single QDs, the surface of which are conjugated with different ligands, such as trioctylphosphine oxide (TOPO), mercaptoundecanoicacid (MDA), and amine modified DNA (AMDNA). In this paper, we present the progress of our measurements of the time-dependent optical characteristics (fluorescence intermittency, photostability, and spectral diffusion) of single MDA-QDs and AMDNA-MDA-QDs in air in an effort to understand the effects of surface-conjugated biomolecules on the optical characteristics at single QD sensitivities.