Recent results on the development and implementation of a novel technology for lung tumor detection and imaging is presented. This technology offers high-sensitivity imaging of magnetic nanoparticles to provide specific diagnostic images of early lung tumors and potential distant metastases. Recent developments in giant magnetostrictive (GMS) or magnetic shape memory (MSM) materials have led to the possibility of developing small, low-cost, room-temperature, portable, high-sensitivity, fiber-optic sensors capable of robustly detecting magnetic nanoparticles, without direct contact with the skin. Magnetic nanoparticles are conjugated with antibodies, which target them to lung tumors. A prototype fiber-optic biomagnetic sensor, based on giant magnetostrictive or magnetic shape memory materials, with the requisite sensitivity to image the magnetic signals generated by antibody-labeled magnetic nanoparticles in lung tumors has been built and calibrated. The uniqueness of the biomagnetic sensor lies in the fact that it offers high sensitivity at room temperature, and is not a SQUID-based system. The results obtained during the process of choosing the right magnetostrictive materials are presented. Then, for the construction of an accurate image of the lung tumor, the optimum spatial distribution of one-channel sensors and nanoparticle polarization has been analyzed.

We describe the design and development two prototype spectroscopy imaging instruments based on custom-made acousto-optic tuneable filters (AOTF). These devices can be coupled to many standard imaging systems (e.g. an endoscope or a fluorescence microscope). The instruments developed offer significant advantages over typical fixed-filter imaging systems in terms of flexibility, performance and diagnostic potential. Any filtering wavelength in the visible range can be rapidly selected either by random access or continuous tuning. Since filtering is achieved through a diffractive process, an excellent signal-to-noise ratio is achieved that allows the detection of extremely low fluorescence signals such as autofluorescence. These adapters were designed to allow the simultaneous imaging of both the filtered and unfiltered signals. A first prototype instrument was developed and demonstrated for in-vivo applications. When attached to the eyepiece of a commercial endoscope, it allowed the simultaneous white light endoscopy and fluorescence imaging. Autofluorescence of protoporphyrin IX (PpIX), an endogenous chromophore that traces early-stage diseased tissue experiencing an inflammatory response, was mapped in vivo on a rat model. The system has also been approved for medical use and human clinical trials are underway. In addition, we are currently testing a second AOTF module for in vitro applications. This new AOTF adapter was designed to be coupled to the viewing port of a commercial fluorescence microscope to realise a fluorescence imaging spectrometer capable of detecting and mapping fluorescent biomolecules.

This paper presents hyperspectral fluorescence imaging and a support vector machine for detecting skin tumors. Skin cancers may not be visually obvious since the visual signature appears as shape distortion rather than discoloration. As a definitive test for cancer diagnosis, skin biopsy requires both trained professionals and significant waiting time. Hyperspectral fluorescence imaging offers an instant, non-invasive diagnostic procedure based on the analysis of the spectral signatures of skin tissue. A hyperspectral image contains spatial information measured at a sequence of individual wavelength across a sufficiently broad spectral band at high-resolution spectrum. Fluorescence is a phenomenon where light is absorbed at a given wavelength and then is normally followed by the emission of light at a longer wavelength. Fluorescence generated by the skin tissue is collected and analyzed to determine whether cancer exists. Oak Ridge National Laboratory developed an endoscopic hyperspectral imaging system capable of fluorescence imaging for skin cancer detection. This hyperspectral imaging system captures hyperspectral images of 21 spectral bands of wavelength ranging from 440 nm to 640 nm. Each band image is spatially co-registered to eliminate the spectral offset caused during the image capture procedure. Image smoothing by means of a local spatial filter with Gaussian kernel increases the classification accuracy and reduces false positives. Experiments show that the SVM classification with spatial filtering achieves high skin tumor detection accuracies.

Early detection of malignant melanoma is critical to improve the survival rates of patients with this aggressive malignancy. We constructed an imaging system employing two liquid-crystal tunable filters to acquire in vivo spectral images of dysplastic lesions from patients at 31 wavelengths from 500 to 950nm. These reflectance images were analyzed in search of optical signatures for quantitative characterization of dysplastic nevi and malignant melanoma. A principal component analysis (PCA) algorithm was developed to examine the spectral imaging data in the component space and an index of spreading of clustering pixels (SCP) was defined to measure the degree of clustering in the distribution of image pixel scores in a component space. We found that SCP of differential polarimetric images correlate strongly with the degree of dysplasia for 4 lesions. However, many questions remain unanswered on the relations between PCA results and the spatial and spectral characteristics of the image data because of limited spectral image data from the patients. To fully improve our understanding on the multivariate analysis of spectral imaging data, we have developed a parallel Monte Carlo code to efficiently generate reflectance images from given distribution of optical parameters in a skin lesion phantom. With this tool, we have investigated numerically the dependence of score distribution and SCP in the component sub-spaces on lesion size and position. These numerical results provide a foundation for our future study to identify optical signature of dysplastic lesion and melanoma in the skin.

Over the last years we have developed a sagittal laser optical tomographic (SLOT) imaging system for the diagnosis and monitoring of inflammatory processes in proximal interphalangeal (PIP) joint of patients with rheumatoid arthritis (RA). While cross sectional images of the distribution of optical properties can now be generated easily, clinical interpretation of these images remains a challenge. In first clinical studies involving 78 finger joints, we compared optical tomographs to ultrasound images and clinical analyses. Receiver-operator curves (ROC) were generated using various image parameters, such as minimum and maximum scattering or absorption coefficients. These studies resulted in specificities and sensitivities in the range of 0.7 to 0.76. Recently, we have trained support vector machines (SVMs) to classify images of healthy and diseased joints. By eliminating redundancy using feature selection, we are achieving sensitivities of 0.72 and specificities up to 1.0. Studies with larger patient groups are necessary to validate these findings; but these initial results support the expectation that SVMs and other machine learning techniques can considerably improve image interpretation analysis in optical tomography.

Objectives: Early detection of cancer and its curable precursors remains the best way to ensure patient survival and quality of life. Despite significant advances in treatment, oral cancer still results in 10,000 U.S. deaths annually, mainly due to the late detection of most oral lesions. Specific aim was to use a combination of non-invasive optical in vivo technologies to test a multi-modality approach to non-invasive diagnostics of oral premalignancy and malignancy. Methods: In the hamster cheek pouch model (120 hamsters), in vivo optical coherence tomography (OCT) and optical Doppler tomography (ODT) mapped epithelial, subepithelial and vascular change throughout carcinogenesis in specific, marked sites. In vivo multi-wavelength multi-photon (MPM) and second harmonic generated (SHG) fluorescence techniques provided parallel data on surface and subsurface tissue structure, specifically collagen presence and structure, cellular presence, and vasculature. Images were diagnosed by 2 blinded, pre-standardized investigators using a standardized scale from 0-6 for all modalities. After sacrifice, histopathological sections were prepared and pathology evaluated on a scale of 0-6. ANOVA techniques compared imaging diagnostics with histopathology. 95% confidence limits of the sensitivity and specificity were established for the diagnostic capability of OCT/ODT+ MPM/SHG using ROC curves and kappa statistics. Results: Imaging data were reproducibly obtained with good accuracy. Carcinogenesis-related structural and vascular changes were clearly visible to tissue depths of 2mm. Sensitivity (OCT/ODT alone: 71-88%; OCT+MPM/SHG: 79-91%) and specificity (OCT alone: 62-83%;OCT+MPM/SHG: 67-90%) compared well with conventional techniques. Conclusions: OCT/ODT and MPM/SHG are promising non-invasive in vivo diagnostic modalities for oral dysplasia and malignancy. Supported by CRFA 30003, CCRP 00-01391V-20235, NIH (LAMMP) RR01192, DOE DE903-91ER 61227, NIH EB-00293 CA91717, NSF BES-86924, AFOSR FA 9550-04-1-0101.

In this study, an x-ray imaging system for small-animal studies was characterized. This system includes an x-ray source with a 20-mm focal spot and a fiber optics coupled CCD detector module. It can be operated in magnification levels ranging from X1.5 to X5, by varying the object-to-detector distance. The system was subjectively characterized using a contrast-detail x-ray phantom. X-ray images of the phantom were obtained and reviewed by observers. The threshold contrast values for different object sizes were determined from observer measurements. The contrast-detail curves showed that magnification reduces the contrast threshold required for object detection. This system is suitable for small animal studies at different magnification levels, with high spatial resolution and adequate contrast-detail detectability.

Real time Monitoring of mitochondrial function in vivo is a significant factor in the understanding of tissue vitality. Nevertheless a single parameter monitoring device is not appropriate and effective in clinical diagnosis of tissue vitality. Therefore we have developed a multi-parametric monitoring system that monitors, in addition to mitochondrial NADH redox state, tissue microcirculatory blood flow, tissue total back-scattered light as an indication of blood volume and blood oxygenation (Hb0₂). In the present communication a new device named "CritiView" is described. This device was developed in order to enable real time monitoring of the four parameters from various organs in the body. The main medical application of the CritiView is in critical care medicine of patients hospitalized in the Intensive Care Units (ICUs) and intraoperatively in operating rooms. The physiological basis for our clinical monitoring approach is based on the well known response to the development of body emergency situation, such as shock or trauma. Under such conditions a process of blood flow redistribution will give preference to vital organs (Brain, Heart) neglecting less vital organs (Skin, G-I tract or the urinary system). Under such condition the brain will by hyperperfused and O₂ supply will increase to provide the need of the activated mitochondria. The non-vital organs will be hypoperfused and mitochondial function will be inhibited leading to energy failure. This differentiation between the two types of organs could be used for the early detection of body deterioration by monitoring of the non-vital organ vitality. A fiber optic sensor was embedded in a Foley catheter, enabling the monitoring of Urethral wall vitality, to serve as an early warning signal of body deterioration.

There have been several attempts to detect bone structural changes using optical techniques. H.G. Eberle et al. have used ultrafast optical techniques on the finger in trans-illumination and have shown an excellent correlation between their measurements of the scattering coefficient and bone mineral density obtained using dual X-ray absorption (DXA) imaging. Encouraged by these results, we have developed a system based on the combination of cw laser light and low-frequency ultrasound to probe the bone structure. The physical principle of this system is the detection of laser light diffusing in the bone tissue modulated ("tagged") by a low-frequency ultrasound pulse, which allows a local measurement of the attenuation coefficient. The basic assumption of the technique is that the main factor of attenuation changes in the bone of elderly patients is a scattering change due to osteoporosis, and therefore attenuation measurements directly reflect the scattering properties of the bone. We present a preliminary series of clinical experiments showing that this technique allows determining the bone scattering modification inside the trabecular bone. In this series of clinical experiments, the scattering coefficient determined using the optical technique is compared with the bone mineral density obtained using dual X-ray absorption in a group of 9 patients. A correlation of 0.84 (p=0.05) was found, showing the potential of this technique for the assessment of osteoporosis.

Aim: In patients, e.g. with congenital heart diseases, a differential blood count is needed for diagnosis. To this end by standard automatic analyzers 500 μl of blood is required from the patients. In case of newborns and infants this is a substantial volume, especially after operations associated with blood loss. Therefore, aim of this study was to develop a method to determine a differential blood picture with a substantially reduced specimen volume. Methods: To generate a differential blood picture 10 μl EDTA blood were mixed with 10 μl of a DRAQ5 solution (500μM, Biostatus) and 10 μl of an antibody mixture (CD45-FITC, CD14-PE, diluted with PBS). 20 μl of this cell suspension was filled into a Neubauer counting chamber. Due to the defined volume of the chamber it is possible to determine the cell count per volume. The trigger for leukocyte counting was set on DRAQ5 signal in order to be able to distinguish nucleated white blood cells from erythrocytes. Different leukocyte subsets could be distinguished due to the used fluorescence labeled antibodies. For erythrocyte counting cell suspension was diluted another 150 times. 20 μl of this dilution was analyzed in a microchamber by LSC with trigger set on forward scatter signal. Results: This method allows a substantial decrease of blood sample volume for generation of a differential blood picture (10 μl instead of 500μl). There was a high correlation between our method and the results of routine laboratory (r²=0.96, p<0.0001; n=40). For all parameters intra-assay variance was less than 7 %. Conclusions: In patients with low blood volume such as neonates and in critically ill infants every effort has to be taken to reduce the blood volume needed for diagnostics. With this method only 2% of standard sample volume is needed to generate a differential blood picture. Costs are below that of routine laboratory. We suggest this method to be established in paediatric cardiology for routine diagnostics and for resource poor settings.

In North America, approximately 30,000 people annually suffer an aneurismal subarachnoid hemorrhage (SAH). Using computerized tomography (CT), the blood is generally not visible after 12 hours. Currently lumbar puncture (LP) results are equivocal for diagnosing SAH largely because of technical limitations in performing a quick and objective evaluation. Having ruptured once, an aneurysm is statistically more likely to rupture again. Therefore, for those individuals with a sentinel (or warning) hemorrhage, detection within the first 12 hours is paramount. We present a diagnostic technology based on visible spectroscopy to quickly and objectively assess low-blood volume SAH from a diagnostic spinal tap. This technology provides clinicians, with the resources necessary for assessing patients with suspected aneurismal SAH beyond the current 12-hour limitation imposed by CT scans. This aids in the improvement of patient care and results in rapid and appropriate treatment of the patient. To perform this diagnosis, we quantify bilirubin and hemoglobin in human CSF over a range of concentrations. Because the bilirubin and hemoglobin spectra overlap quantification is problematic. To solve this problem, two algorithmic approaches are presented: a statistical or a random stochastic component known as Partial Least Square (PLS) and a control theory based mathematical model. These algorithms account for the noise and distortion from blood in CSF leading to the quantification of bilirubin and methemoglobin spectroscopically. The configurations for a hardware platform is introduced, that is portable and user-friendly composed of specific components designed to have the sensitivity and specificity required. This aids in measuring bilirubin in CSF, hemorrhagic-CSF and CSF-like solutions. The prototype uses purpose built algorithms contained within the platform, such that physicians can use it in the hospital and lab as a point of care diagnostic test.

Fluorescence spectroscopy plays a key role in a broad area of biological and medical applications. Development of fluorescence spectroscopy micro-devices will enable construction of fully integrated platforms for clinical diagnostics. We report the design, microfabrication and testing of a piezoelectric MEMS micro-grating as a part of the development of a combined spectral/time-resolved fluorescence biosensor for tissue characterization. For the design of the device, we simulated its theoretical performance using a piezoelectric multi-morph model with appropriate diffraction geometry. The microfabrication process was based on a SiN diaphragm (formed via KOH bulk-micromachining) on which the supporting layer of the micro-cantilevers was patterned. Piezoelectric ZnO was then magnetron sputtered and patterned on the cantilever as the physical source for linear actuation with low voltage (>32V). E-beam evaporation of aluminum formed the final reflective diffraction pattern as well as the electrode connections to the device units. The device actuation and displacement were characterized using LDDM (Laser Doppler Displacement Meter). Current cantilevers designed with 500 μm wide gratings (20 μm spacing) produced a maximum 38 μm bi-polar deflection at 3.5 kHz, with scanning from 350-650 nm at 26 nm resolution (10 nm with new 10 μm period prototype). The MEMS device was designed to be integrated with a fast response photomultiplier, and thus can be used with time-resolved fluorescence detection. Because in the case of time-resolved measurements, spectral resolution is not a crucial element, this configuration allows for the compensation of the geometric limitations (linear dispersion) of a micro-scale device that require wavelength differentiation and selection.

A setup to measure skin autofluorescence was developed to assess accumulation of advanced glycation endproducts (AGE) in patients noninvasively. The method applies direct blacklight tube illumination of the skin of the lower arm, and spectrometry. The setup displays skin autofluorescence (AF) as a ratio of mean intensities detected from the skin between 420-600 nm and 300-420 nm, respectively. In an early clinical application in 46 and control subjects matched for age and gender, AF was significantly increased in the patients (p = 0.015), and highly correlated with skin AGE's that were determined from skin biopsies in both groups. A large follow-up study on type 2 diabetes mellitus, ongoing since 2001 with more than 1000 subjects, aims to assess the value of the instrument in predicting chronic complications of diabetes. At baseline, a relation with age, glycemic status and with complications present was found. In a study in patients with end stage renal disease on dialysis AF was a strong and independent predictor of total and cardiovascular mortality. A commercial version of this AGE-reader is now under development and becomes available early 2005 (DiagnOptics B.V., Groningen, The Netherlands). One of the remaining questions, that will be answered by measuring so-called Exciation-Emission Matrices (EEM's) of the skin tissue in vivo, is whether a more selective choice of wavelengths is more strongly related to clinical characteristics. An experimental instrument to measure these EEM's was, therefore, developed as well. Clinical measurements are underway of EEM's in patient groups with diabetes mellitus and in healthy volunteers.

A time-resolved 3-channel data acquisition system is designed to study the temporal characteristics of acetowhitening at cellular level. Both normal and cancerous cells from the ectocervical tissue are studied and the intensity of the backscattering light from the monolayer cells is recorded and analyzed. It is found that the intensity decay courses of normal and cancerous cells are quite different in the line shape. Double-exponential decay model is used to fit the curves and the calculated time constant is used to quantitatively distinguish the normal and cancerous cells. The time constant of cancerous cells is longer than that of normal cells when the same concentration of acetic acid is used. The study shows the potential of this method to distinguish normal and cancerous tissues from the decay course of acetowhitening. The quantification of the acetowhitening effect could be potentially used for the objective detection of neoplastic lesions at cervical tissue.

The depth-resolved autofluorescence of normal and dysplastic human ectocervical tissue within 120um depth were investigated utilizing a portable confocal fluorescence spectroscopy with the excitations at 355nm and 457nm. From the topmost keratinizing layer of all ectocervical tissue samples, strong keratin fluorescence with the spectral characteristics similar to collagen was observed, which created serious interference in seeking the correlation between tissue fluorescence and tissue pathology. While from the underlying non-keratinizing epithelial layer, the measured NADH fluorescence induced by 355nm excitation and FAD fluorescence induced by 457nm excitation were strongly correlated to the tissue pathology. The ratios between NADH over FAD fluorescence increased statistically in the CIN epithelial relative to the normal and HPV epithelia, which indicated increased metabolic activity in precancerous tissue. This study demonstrates that the depth-resolved fluorescence spectroscopy can reveal fine structural information on epithelial tissue and potentially provide more accurate diagnostic information for determining tissue pathology.

We present the principles and applications of our dual-modality fluorescence and reflectance hyperspectral imaging (DMHSI) system. In this paper we report on background work done using laser induced fluorescence (LIF) by the group in the early detection of esophageal cancer. We then demonstrate the capabilities of our new DMHSI system. The system consists of a laser, endoscope, AOTF, and two cameras coupled with optics and electronics. Preliminary results, performed on mouse tissue, show that the system can delineate normal and malignant tissue regions in real-time.

Patients with Barrett's esophagus (BE) undergo periodic endoscopic surveillance with random biopsies in an effort to detect dysplastic or early cancerous lesions. Surveillance may be enhanced by near-infrared Raman spectroscopy (NIRS), which has the potential to identify endoscopically-occult dysplastic lesions within the Barrett's segment and allow for targeted biopsies. The aim of this study was to assess the diagnostic performance of NIRS for identifying dysplastic lesions in BE in vivo. Raman spectra (P_exc=70 mW; t=5 s) were collected from Barrett's mucosa at endoscopy using a custom-built NIRS system (λ_exc=785 nm) equipped with a filtered fiber-optic probe. Each probed site was biopsied for matching histological diagnosis as assessed by an expert pathologist. Diagnostic algorithms were developed using genetic algorithm-based feature selection and linear discriminant analysis, and classification was performed on all spectra with a bootstrap-based cross-validation scheme. The analysis comprised 192 samples (112 non-dysplastic, 54 low-grade dysplasia and 26 high-grade dysplasia/early adenocarcinoma) from 65 patients. Compared with histology, NIRS differentiated dysplastic from non-dysplastic Barrett's samples with 86% sensitivity, 88% specificity and 87% accuracy. NIRS identified 'high-risk' lesions (high-grade dysplasia/early adenocarcinoma) with 88% sensitivity, 89% specificity and 89% accuracy. In the present study, NIRS classified Barrett's epithelia with high and clinically-useful diagnostic accuracy.

In this study FT-RAMAN spectra of breast tissue from 35 patients were obtained and separated into nine groups for histopathologic analysis, which are as follows: normal breast tissue, fibrocystic condition, in situ ductal carcinoma, in situ ductal carcinoma with necrosis, infiltrate ductal carcinoma, infiltrate inflammatory ductal carcinoma, infiltrate medullar ductal carcinoma, infiltrate colloid ductal carcinoma, and infiltrate lobular carcinoma. Using spectrum averages taken from each group a qualitative analysis was performed to compare these molecular compositions to those known to be present in abnormal concentrations in pathological situations, e.g. the development of desmoplastic lesions with a stroma of dense collagen in tumoral breast tissues which substitute adipose stroma of non-diseased breast tissue. The band identified as amino acids, offered basis for observation in the existence of alterations in the proteins, thus proving Raman Spectroscopic capacity in identification of primary structures of proteins; secondary protein structure was also identified through the peptic links, Amide I and Amide III, which have also been identified by various authors. Alterations were also identified in the peaks and bandwidths of nucleic acids demonstrating the utilization of Raman Spectroscopy in the analysis of the cells nucleus manifestations. All studies involving Raman Spectroscopy and breast cancer have shown excellent result reliability and therefore a basis for the technical theory.

Imaging polarimetry is presently used in many different biomedical applications, like ophthalmology or dermatology, providing a non-invasive, non-contact and high-resolution diagnosis. Polarization techniques are based on the analysis of biological tissues by means of the Mueller matrix, which provides a complete description of the polarization properties, but this matrix does not supply a clear and comprehensive information about the physical and biological properties of tissues at first glance, and a slow and meticulous analysis of the matrix elements has to be accomplished to obtain a detailed characterization of their behavior. In this work, the entropy factor is defined and introduced for the analysis of biological tissues in order to handle one single parameter whose value is closely related to the tissue behavior, and therefore, provides a clearer analysis of the biological and physical modifications that take place in an unhealthy, old or heat-damaged tissue through its polarization characteristics. The entropy factor will be applied to the analysis of the Mueller matrices of different biological tissues obtained. Specifically, it is utilized for the characterization of different tissues, porcine tendon and rat tail, which has been analyzed in two situations, normal and burned, to observe if the entropy factor suffers a variation. This study is performed to provide a confident instrument to medical staff in biological and medical examination of tissues, so that a medical diagnosis can be realized through the study of this factor, providing in the next future an extra instrument for a complete development of optical biopsies by means of polarization techniques.

The human cardiovascular system is a complex system with the pumping activity of the heart as the main generator of oscillations. Besides the heartbeat there are several other oscillatory components which determine its dynamics. Their nonlinear nature and a weak coupling between them both require special treatment while studying this system. A particular characteristic of the oscillatory components is their frequency fluctuations in time. Consequently, their interactions also fluctuate in time. Therefore the wavelet transform is applied to trace the oscillatory components in time, and specific quantitative measures are introduced to quantify the contribution of each of the oscillatory components involved on the time scale of up to three minutes. Oscillatory components are then analysed from signals obtained by simultaneous measurements of blood flow in the microcirculation, ECG, respiration and blood pressure. Based on quantitative evaluation of the oscillatory components related to (I) the heart beat (0.6-2Hz), (II) respiration (0.145-0.6Hz), (III) intrinsic myogenic activity (0.052-0.145Hz), (IV) sympathetic activity (0.021-0.052Hz), (V, VI) endothelial related activity (0.0095-0.021Hz, 0.005 - 0.0095 Hz), 30-minutes recording taken on 109 healthy subjects, 75 patients with diabetes, and 82 patients after acute myocardial infarction (AMI) were analysed. Classification of the effect of ageing, diabetes and AMI from blood flow signals simultaneously recorded in the skin of four extremities, the heart rate and heart rate variability from R-R intervals will be presented and discussed.

Microfluidic devices are becoming increasingly popular for many applications, enabling biological and chemical reactions to be performed with nano- and picoliter sample volumes. Accurate measurement and monitoring of fluid flow behavior in the small channels of microfluidic systems is important for evaluating the performance of existing devices, and in the modeling and design of new microfluidic networks. We present here the results of experiments using spectral-domain optical Doppler tomography (SD-ODT) to measure fluid flow in single-layer microfluidic devices. The principles behind flow imaging with SD-ODT are reviewed, a method for velocity calibration is described, and cross-sectional and en-face images of fluid velocity in microfluidic channels are presented.

Stroke remains the third most frequent cause of death in the United States and the leading cause of disability in adults. Long-term effects of ischemic stroke can be mitigated by the opportune administration of Tissue Plasminogen Activator (t-PA); however, the decision regarding the appropriate use of this therapy is dependant on timely, effective neurological assessment by a trained specialist. The lack of available stroke expertise is a key barrier preventing frequent use of t-PA. We report here on the development of a prototype research system capable of performing a semi-automated neurological examination from an offsite location via the Internet and a Computed Tomography (CT) scanner to facilitate the diagnosis and treatment of acute stroke. The Video Stroke Assessment (VSA) System consists of a video camera, a camera mounting frame, and a computer with software and algorithms to collect, interpret, and store patient neurological responses to stimuli. The video camera is mounted on a mobility track in front of the patient; camera direction and zoom are remotely controlled on a graphical user interface (GUI) by the specialist. The VSA System also performs a partially-autonomous examination based on the NIH Stroke Scale (NIHSS). Various response data indicative of stroke are recorded, analyzed and transmitted in real time to the specialist. The VSA provides unbiased, quantitative results for most categories of the NIHSS along with video and audio playback to assist in accurate diagnosis. The system archives the complete exam and results.

The dynamical characteristics of integral mechanical properties of drying droplets of blood serum, urine and saliva were studied by measuring the acoustic-mechanical impedance (AMI) using a computer-controlled setup described earlier. The method is based on the following idea. When a 5μl liquid drop is drying on the surface of a quartz resonator plate oscillating at a constant frequency (equal to the resonance frequency of an unloaded resonator, 60 kHz), there arises a shear wave highly sensitive to the formation and growth of a new phase at the liquid-quartz interface. The experimental setup measures a change in the complex electric conductivity of the liquid-quartz system, calculates the parameters of the AMI dynamics in the drying drop, and displays their variation on the monitor at the real time scale in the form of a curve. At a time the optical The properties of drying drops were observed. For each disease or a physiological state the geometrical features of the curves were extracted and then the shape indices were calculated. In the cases under study each "pathology" group differed from the "norm" by its specific shape index, by which diseases of other nature did not differ. Possible mechanisms behind formation of the morphological and dynamical differences in drying drops of biological liquids of healthy and sick people are discussed.

Nanomedicine involves cell-by-cell regenerative medicine, either repairing cells one at a time or triggering apoptotic pathways in cells that are not repairable. Multilayered nanoparticle systems are being constructed for the targeted delivery of gene therapy to single cells. Cleavable shells containing targeting, biosensing, and gene therapeutic molecules are being constructed to direct nanoparticles to desired intracellular targets. Therapeutic gene sequences are controlled by biosensor-activated control switches to provide the proper amount of gene therapy on a single cell basis. The central idea is to set up gene therapy "nanofactories" inside single living cells. Molecular biosensors linked to these genes control their expression. Gene delivery is started in response to a biosensor detected problem; gene delivery is halted when the cell response indicates that more gene therapy is not needed. Cell targeting of nanoparticles, both nanocrystals and nanocapsules, has been tested by a combination of fluorescent tracking dyes, fluorescence microscopy and flow cytometry. Intracellular targeting has been tested by confocal microscopy. Successful gene delivery has been visualized by use of GFP reporter sequences. DNA tethering techniques were used to increase the level of expression of these genes. Integrated nanomedical systems are being designed, constructed, and tested in-vitro, ex-vivo, and in small animals. While still in its infancy, nanomedicine represents a paradigm shift in thinking-from destruction of injured cells by surgery, radiation, chemotherapy to cell-by-cell repair within an organ and destruction of non-repairable cells by natural apoptosis.

Nanoparticles are increasingly finding a wide application in the biological studies due to their unique physical and chemical properties. Colloidal nanoparticles are efficient substrate that exhibit surface-enhanced Raman scattering (SERS) phenomenon by enhancing the scattering cross-sections of conjugated Raman active molecules thus enabling highly sensitive biological probes. However, biological and medical applications would require nanoparticles to be conjugated to biomolecules. A universal approach for conjugation of mercarptoacetic acid-capped silver nanoparticles to biomolecules is described. The surface functionalized silver colloids were labeled with a Raman active dye and used for cellular imaging. We also described the use of silver nanoparticle to develop a new class of SERS nanoprobes for molecular recognition and detection of specific nucleic acid sequences.

For proteomics drug discovery applications, combinatorial microbead thioaptamer libraries (one thioaptamer sequence per bead) are being created by split synthesis method, creating a "proteomics library" of protein capture beads which can be analyzed by high-throughput screening methods in this case, flow cytometry and cell sorting. Thioaptamers, oligonucleotides with thiophosphate backbone substitutions, function like antibodies in terms of recognizing specific protein sequences but have a number of advantages over antibody libraries. These proteomics beads can then be analyzed by high-speed flow cytometry and sorted to single-bead level depending on relative fluorescence brightness of fluorescently-labeled proteins, or for a specific protein from all of the molecules of cell subpopulations being analyzed. The thioaptamer sequences on a given bead showing high affinity for that protein can then be sequenced. Alternatively, the protein-capturing beads can be analyzed by MALDI-TOF mass spectrometry for analysis of the bound proteins. The beads can be thought of as equivalent to single-element positions of a proteomics chip arrays but with the advantage of being able to much more rapidly analyze hundreds of millions of possible amino acid sequences/epitopes on the basis of thioaptamer sequence affinities to select single sequences of interest. Additionally, those beads can be manipulated and isolated at the single bead level by high-throughput flow cytometry/cell sorting for subsequent sequencing of the thioaptamer sequences.

The speed of interferometric detection is at least 1000 times faster than the fluorometric detection used in the vast majority of clinical diagnostic systems. This opens the possibility to perform thousands of assays in the time it takes fluorescence to perform only one. Molecules immobilized on a spinning disk, like a CD, present the fastest and simplest means of interrogating thousands of micron-scale interferometer elements per second. However, the challenge of interferometry on a spinning disk is to maintain stable phase in the presence of mechanical vibration. In this paper, we demonstrate the first use of adaptive optics in an adaptive optical homodyne mixer to perform interferometry on a multi-analyte BioCD. The BioCD is a 4" diameter glass disk printed with a spoke pattern of protein. When the disk spins, the periodic protein pattern is transferred into a high-speed optical phase modulation by spinning the disk at 3000 rpm in the path of a probe laser. A nonlinear optical film mixes the signal beam with a stable reference beam in a two-wave mixing configuration that adaptively phase-locks the two beams to create stable phase in spite of mechanical vibration. Specific binding of antibody to printed antigen is detected as an increased homodyne signal. Multi-analyte detection on Anti Mouse and Anti Rabbit IgG is performed in which Mouse IgG and Rabbit IgG act as the non-specific reagent to each other. Detection is made on circular tracks. The technique has the potential of fast screening for large numbers of protein interactions.

This work presents the first demonstration of the characterization of cancer cells using Fourier Transform Infrared Spectroscopy (FTIR) technique in the low terahertz frequency range of 10-25 cm^-1 and shows how this technique can be used to discriminate between two types of cancer (prostate and bladder) in cell culture. Samples of cancer cells suspended in buffer solution with the ratio of dry material to liquid ~ 1:10 were prepared and measured. The work will serve as the foundation for future work defining specific spectra associated with different tumor histologies and tumor progression and metastasis.

Terahertz pulsed imaging is a non-invasive, non-ionizing imaging technique, using electromagnetic radiation defined in the frequency range 0.1 THz to 10 THz. Using reflection imaging systems with a frequency range 0.1 THz to 4 THz a far field diffraction limited lateral resolution of 3 mm to 110 microns is attainable. A three layer analytical model has been developed to simulate the hydration properties of skin in reflection. Earlier in vivo hydration measurements of the volar forearm and palm of the hand (thenar) are compared to this model. The time-domain analysis technique “time post pulse” (TPP) used to differentiate between diseased and normal tissue in a study of basal cell carcinoma was applied to the data. An increase in the value of TPP is observed with occlusion in the viable epidermis. This is attributed to an increase in the flux of water across the epidermis or dermis with increased stratum corneum hydration. This is verified by the literature. The change is observed in less than six minutes occlusion, making terahertz technology one of the most sensitive techniques for monitoring skin hydration levels. The contrast observed at the stratum corneum-viable epidermis interface is similar to that seen between diseased and normal tissue. Although water provides a good marker for studying diseased tissue, comparing results from DNA and protein analysis, it is not yet possible to conclude whether the contrast observed in basal cell carcinoma is due to increased water within the diseased tissue, a change in the vibrational modes of water with other functional groups, or a change in the vibrational modes of the functional groups alone. Further studies are required to determine whether terahertz technology is capable of differentiating between different histological subtypes in a collective system such as skin at a macroscopic level. The three layer analytical model provides a useful adjunct for identifying the source of contrast observed in the top surface of skin.

Terahertz imaging has shown great potential in several biomedical areas such as burn imaging, detection of skin cancer, and pharmaceutical tablet imaging. The development of each of these application has been limited by slow imaging speed (tens of minutes to hours) and small scan areas (less than 10 square centimeters). Elsewhere to date, the sample itself must be mechanically raster scanned due to the free space optical coupling of femtosecond laser pulses driving the terahertz generating and detecting elements. This paper reports on the development of a freely positionable fiber optic coupled terahertz transceiver which may be raster scanned over a stationary object. Image acquisition times of less than 8 minutes for a 20x20 cm area (400 sq cm area) raster scanning a terahertz transceiver over a stationary object; and of less than 1 minute for image acquisition with a movable object raster scanning the object have been demonstrated. High speed stationary imaging will allow the practical investigation on human and animal subjects.

Nanosecond, high intensity pulsed electric fields [nsPEFs] that are below the plasma membrane [PM] charging time constant have decreasing effects on the PM and increasing effects on intracellular structures and functions as the pulse duration decreases. When human cell suspensions were exposed to nsPEFs where the electric fields were sufficiently intense [10-300ns, ≤300 kV/cm.], apoptosis signaling pathways could be activated in several cell models. Multiple apoptosis markers were observed in Jurkat, HL-60, 3T3L1-preadipocytes, and isolated rat adipocytes including decreased cell size and number, caspase activation, DNA fragmentation, and/or cytochrome c release into the cytoplasm. Phosphatidylserine externalization was observed as a biological response to nsPEFs in 3T3-L1 preadipocytes and p53-wildtype and -null human colon carcinoma cells. B10.2 mouse fibrosarcoma tumors that were exposed to nsPEFs ex vivo and in vivo exhibited DNA fragmentation, elevated caspase activity, and reduced size and weight compared to contralateral sham-treated control tumors. When nsPEF conditions were below thresholds for apoptosis and classical PM electroporation, non-apoptotic responses were observed similar to those initiated through PM purinergic receptors in HL-60 cells and thrombin in human platelets. These included Ca²⁺ mobilization from intracellular stores [endoplasmic reticulum] and subsequently through store-operated Ca²⁺ channels in the PM. In addition, platelet activation measured as aggregation responses were observed in human platelets. Finally, when nsPEF conditions followed classical electroporation-mediated transfection, the expression intensity and number of GFP-expressing cells were enhanced above cells exposed to electroporation conditions alone. These studies demonstrate that application of nsPEFs to cells or tissues can modulate cell-signaling mechanisms with possible applications as a new basic science tool, cancer treatment, wound healing, and gene therapy.

The charging of mammalian cell plasma membranes in response to ultrashort pulsed electric fields of 60 ns and field strengths up to 100 kV/cm was investigated. Membranes of Jurkat cells were stained with a potential-sensitive dye, Annine-6 and placed in a microreactor mounted on an inverted fluorescence microscope. Images of changes in the fluorescence intensity during the exposure were recorded with a high-sensitivity CCD-camera. A temporal resolution of 5 ns was achieved by illuminating the cells with a 5 ns laser pulse from a dye-laser. The laser pulse was synchronized with the high voltage pulse to record images at specific times before, during and after exposure to the electric field. When exposing Jurkat cells to a 60 ns, 100 kV/cm pulse, each hemisphere of the plasma membrane (as oriented with respect to the electrodes) responded uniquely to the applied field. From these observations it is possible to draw conclusions on the charging time of the membrane, maximum transmembrane voltages and the onset of poration.

This paper reports preliminary results from the development and application of a two-dimensional MEMS endoscopic scanner for OCT imaging. A 1 mm diameter mirror provides high aperture over large scan angle and can scan at rates of hundreds of Hz in both axes. The mirror is integrated with focusing optics and a fiber-optic collimator into a package of ~5 mm diameter. Using a broadband femtosecond laser light source, ultrahigh axial image resolution of < 5 um in tissue is achieved at 1.06 um center wavelength. Ultrahigh resolution cross-sectional and three-dimensional OCT imaging is demonstrated with the endoscope with ~12 um transverse resolution and < 5 um axial resolution.

Optical Coherence Microscopy (OCM) enables the acquisition of high resolution, en face images. Most current OCM systems are based on slow analog or high speed digital demodulation schemes. In this paper we demonstrate a low-cost, high speed analog fringe generation and demodulation method. A high power operational amplifier drives a mirrored piezoelectric stack mounted in the reference arm of the interferometer. The drive signal is synchronized with the demodulation frequency of two analog lock-in amplifiers, which extract the first and second harmonic power of the coherence fringes. Tenth order Bessel low-pass filters (LPFs) allow fast system response and reduce carrier frequency noise. Four outputs (X and Y components of first and second harmonic) are acquired with a low-cost data acquisition board and combined to eliminate the slow phase drift in the interferometer. C# software processes and displays the image, and performs automatic interferometer pathlength matching and adjustment. We present images of Arabidopsis leaf in situ, sections of carrot, and ex vivo rat ovary. Excellent image quality is achieved at acquisition speeds up to 40,000 samples/second.

Optical Coherence Tomography (OCT) and Laser Induced Fluorescence Spectroscopy (LIF) have separately been found to have clinical potential in identifying human gastrointestinal (GI) pathologies, yet their diagnostic capability in mouse models of human disease is unknown. We combine the two modalities to survey the GI tract of a variety of mouse strains and sample dysplasias and inflammatory bowel disease (IBD) of the small and large intestine. Segments of duodenum and lower colon 2.5 cm in length and the entire esophagus from 10 mice each of two colon cancer models (ApcMin and AOM treated A/J) and two IBD models (Il-2 and Il-10) and 5 mice each of their respective controls were excised. OCT images and LIF spectra were obtained simultaneously from each tissue sample within 1 hour of extraction. Histology was used to classify tissue regions as normal, Peyer’s patch, dysplasia, adenoma, or IBD. Features in corresponding regions of OCT images were analyzed. Spectra from each of these categories were averaged and compared via the student's t-test. Features in OCT images correlated to histology in both normal and diseased tissue samples. In the diseased samples, OCT was able to identify early stages of mild colitis and dysplasia. In the sample of IBD, the LIF spectra displayed unique peaks at 635nm and 670nm, which were attributed to increased porphyrin production in the proliferating bacteria of the disease. These peaks have the potential to act as a diagnostic for IBD. OCT and LIF appear to be useful and complementary modalities for imaging mouse models.

Mouse models are increasingly important for studying human GI pathology. OCT provides minimally invasive, cross-sectional images that indicate the thickness and scattering density of underlying tissue. We have developed endoscopic ultrahigh resolution OCT (UHR-OCT) for the purpose of in vivo imaging in mouse colon. The reduced scale of the mouse colon makes tissue light penetration much less problematic, and high resolution acutely necessary. Higher lateral resolution requires a departure from the traditional cemented GRIN lens design. We support the need for better chromatic aberration than can be achieved by a GRIN lens using commercial raytracing software. We have designed and built a 2mm diameter endoscopic UHR-OCT system achromatized for 770-1020nm for use with a Titanium:sapphire laser with 260 nm bandwidth at full-width-half-maximum centered at 800 nm while achieving a 4.4um lateral spot dimension at focus. A pair of KZFSN5/SFPL53 doublets provides excellent primary and secondary color correction to maintain wide bandwidth through the imaging depth. A slight deviation from normal beam exit angle suppresses collection of the strong back reflection at the exit window surface. The novel design endoscope was built and characterized for through focus bandwidth, axial resolution, signal to noise, and lateral spot dimension. Performance is demonstrated on a variety of ex vivo tissues and in situ mouse colon. Ultrahigh-resolution images of mouse tissue enable the visualization of microscopic features, including crypts that have previously been observed with standard resolution OCT in humans but were too small to see in mouse tissue. Resolution near the cellular level is potentially capable of identifying abnormal crypt formation and dysplastic cellular organization.

We present a new method for multi-color fluoroimmunoassays based on directional surface plasmon-coupled emission (SPCE). SPCE is coupling of excited fluorophores with a nearby thin metal film (silver) resulting in strongly directional emission into the underlying glass substrate. The angle at which the radiation propagates through the prism depends on emission wavelength and makes possible measurement of multiple analytes using multiple emission wavelengths. We demonstrated this possibility using two antibodies labeled with different fluorophores, binding to an antigen protein immobilized on the silver surface. We observed independent emission at a different angle on the glass prism, resulting of the surface binding of each antibody. This methodology can be readily extended to 3 or more fluorophores. This technology presents opportunity to develop highly sensitive multiplex assay format for biological agents' detection.

Light transmission measurements in the wavelength range of 400 to 1000 nm were performed on Bacillus subtilis spores at periodic time intervals after heat-shock induced activation. The Gaussian ray approximation, using a concentric sphere model for the spore coat and spore core, was used to calculate the scattering cross-section of the spores. Analysis of transmission spectra determined that the refractive index of the spore core was 1.515. In the three hours following heat shock, the core refractive index decreased to 1.39, and subsequently remained constant. During the measurements, the spore radius increased from 0.38 microns to 0.6 microns. The results were confirmed by phase contrast microscopy.

This paper describes a compact, self-contained, cost effective, and portable Raman Integrated Tunable Sensor (RAMiTs) for screening a wide variety of chemical and biological agents for homeland defense applications. The instrument is a fully-integrated, tunable, "point-and-shoot" Raman monitor based on solid-state acousto-optic tunable filter (AOTF) technology. It can provide direct identification and quantitative analysis of chemical and biological samples in a few seconds under field conditions. It also consists of a 830-nm diode laser for excitation, and an avalanche photodiode for detection. Evaluation of this instrument has been performed by analyzing several standard samples and comparing the results those obtained using a conventional Raman system. In addition to system evaluation, this paper will also discuss potential applications of the RAMiTs for detection of chemical and biological warfare agents.

Fluorescence and absorption spectroscopy were performed on Bacillus subtilis spores which were heat treated at temperatures ranging from 20°C to 90°C. The tryptophan (trp) emission from the spore suspensions treated at temperatures greater than 60°C was shifted towards longer wavelength as compared to the spores which were heat treated at lower temperatures. These spectral changes were the result of proteins released by the spores into the suspension. Trp residues in the emitted proteins are in a more polar environment and therefore exhibit a larger Stokes shift. Fluorescence and absorption measurements show that the concentration of proteins in the supernatant was greater for spores treated at higher temperatures. Electrophoresis gel analysis showed the presence of a 47 KDa protein in the supernatant.

Contamination of drinking water with pathogenic microorganisms such as Cryptosporidium has become an increasing concern in recent years. Cryptosporidium oocysts are particularly problematic, as infections caused by this organism can be life threatening in immunocompromised patients. Current methods for monitoring and analyzing water are often laborious and require experts to conduct. In addition, many of the techniques require very specific reagents to be employed. These factors add considerable cost and time to the analytical process. Raman spectroscopy provides specific molecular information on samples, and offers advantages of speed, sensitivity and low cost over current methods of water monitoring. Raman spectroscopy is an optical method that has demonstrated the capability to identify and differentiate microorganisms at the species and strain levels. In addition, this technique has exhibited sensitivities down to the single organism detection limit. We have employed Raman spectroscopy and Raman Chemical Imaging, in conjunction with chemometric techniques, to detect small numbers of oocysts in the presence of interferents derived from real-world water samples. Our investigations have also indicated that Raman Chemical Imaging may provide chemical and physiological information about an oocyst sample which complements information provided by the traditional methods. This work provides evidence that Raman imaging is a useful technique for consideration in the water quality industry.

A novel structural architecture of the substrate for Surface Enhanced Raman Spectroscopy utilizes the effect of light localization in macroporous material and results in significant improvement of low-concentrated detection of contaminants in solution.

Detecting biological particles and subsequently identifying them in a very short period of time is highly desirable, but a very difficult task. There are several pathways for developing rapid detection systems. For example, one can reduce sample size to a very small volume, and amplify cellular components by PCR technology with a view to identifying antigen-specific molecules. Alternatively, antibody-based assays allow for detection and identification of a variety of well-characterized pathogens. The system we propose utilizes flow cytometry technology to rapidly detect spectral fingerprints or organisms. However, the current limit for simultaneously detectable fluorescence signals in flow cytometry is around 12-15. Making these measurements is very complex and the necessity for advanced spectral overlap calculations creates a number of difficult problems to solve in a short period of time. Next-generation instruments can either increase the number of detectors or modify the principles of collection. If the detector system were simplified, the overall cost and complexity of single-cell analytical systems might be reduced. This requires changes in both hardware and software that allow for the analysis of 30 or more spectral signals. Further, analysis of complex data sets requires some completely new approaches, particularly in the area of multispectral analysis. This presentation describes the key components and principles involved in building a next-generation instrument which can collect simultaneously 32 bands of fluorescence from a particle in less than 5 microseconds. This would allow the analysis of several thousand bioparticles per second. The flow cytometry system based on our new detector would be designed to be portable and low cost.

A multi-analyte multi-color immunoassay utilizing enhanced fluorophores fluorescence in the proximity of silver island film (SIF) coated surface is described. A mixture of four different model analyte proteins was immobilized on the surface, and fluorescence signal of four corresponding antibodies labeled with different color fluorophores (emission maxima varied from 570 to 700 nm) was monitored using appropriate cut-off filters. To localize the excitation volume within close proximity of the SIF surface we used the total internal reflection (TIR) mode of excitation. Use of SIF coating resulted in approximately 5-10 fold increase in the intensity of the fluorophores signal if compared to the non-coated glass substrate surface. We suggest application of this methodology for multiplex immunoassays for simultaneous detection of several different analytes in medicine and biotechnology.

Using a near-infrared optical device developed by ViOptix, Inc., a clinical study on post-operative non-invasive monitoring of finger blood perfusion has been conducted for 48 patients undergoing digital replantation at the California Pacific Medical Center. The study showed that non-survival digits have their tissue oxygen saturation (StO₂) values significantly lower than those for the controls in general, but survival digits did not. Further, the StO₂ values can be used to define a survival index, in terms of which a digit survival criterion was tentatively suggested. Applying the criterion to the 64 digits (with 3 of them non-survival) involved in the clinical study, the sensitivity and the specificity were high. Therefore the device may have potential to be used in post-operative monitoring for digit replantation.

Zinc ion is of growing interest in medicine and biology generally, and especially in the ischemic brain and other tissues. We have developed ratiometric fluorescence-based biosensors for the study of zinc in these systems; the biosensors use apocarbonic anhydrase variants as recognition elements that offer high sensitivity and selectivity. We report continuous in situ, in vivo measurement of nanomolar extracellular zinc in the brain of an animal model of ischemia using a ratiometric fiber optic biosensor. We also report the development of an expressible excitation ratiometric indicator of zinc ion suitable for use in cells that exhibits picomolar sensitivity. Finally, we also report the discovery that the Zn complex of the chelator TPEN seems to be comparably apoptogenic to the free chelator itself.