A noninvasive blood glucose monitoring device will provide an invaluable tool in the diagnosis and treatment of diabetes. Near infrared (NIR) absorption spectroscopy is one of the most promising optical techniques for in vivo blood glucose sensing to date. Successful realization of such a technology hinges on solving two main problems. First, instrument sensitivity needs to be improved in order to resolve the weak NIR spectral variations due to glucose physiological changes in the blood. Second, interfering signals due to other blood components and tissue changes need to be sufficiently eliminated or compensated for. A simple, low-cost, high-throughput, filter spectrometer optimized for long-wave NIR measurements of biological fluids is developed. The instrument provides noise spectra with a typical rms value of 7 μAU between 2180 nm and 2310 nm with only 5 seconds of data measurement or averaging. Using such an instrument, spectra of aquaeous, synthetic biological solutions containing varying levels of glucose, BSA, triacetin, lactate and urea are obtained. Glucose spectra are isolated, despite the overlapping spectra. Glucose concentrations are predicted with excellent accuracy (SEP≤8.2 mg/dL) using the simple classical least-squares (CLS) and the connonly used partial least-squares (PLS) multivariate techniques.

We have performed in vivo measurements of near-infrared rat skin absorption in the 4000-5000 cm^-1 spectral range (2.0-2.5 μm wavelength) during a glucose clamp experiment. The goal of this work is to identify the presence of glucose-specific spectral information in order to evaluate the requirements for a noninvasive transcutaneous glucose instrument. Skin spectra are collected using an FTIR spectrometer coupled with a fiber-optic interface. In the experiment, an animal is allowed to stabilize at a euglycemic level for three hours while blood glucose values are monitored using samples taken from an arterial catheter. The blood glucose level is then increased above 30 mM by venous infusion of glucose and held for two hours, after which it is allowed to return to normal. Spectra are recorded continuously during the procedure and are analyzed to identify changes due to the glucose variations. Because the change in absorbance due to an increase in glucose concentration is small compared to changes due to other variations (e.g., the thickness of the skin sample), a simple subtraction of absorbance spectra from the hyperglycemic and euglycemic phases is not instructive. Instead, a set of principal components is determined from the euglycemic period where the glucose concentration is constant. We then examine the change in absorbance during the hyperglycemic period that is orthogonal to these principal components. We find that there are significant similarities between these orthogonal variations and the net analyte signal of glucose, which suggests that glucose spectral information is present.

Minimally-invasive glucose sensors are attractive for disease management as well as medical and biological research applications. Fluorescent sensors, coupling enzymes to catalyze specific reactions with glucose, have been developed for this purpose. Our work has focused on the downscaling of these technologies to the micro- and nano-scale by using self-assembly to build the sensing components of the assays. Both "solid" polymers and hollow microshells have been developed to physically couple the sensing materials together in biocompatible, semipermeable packages suitable for use in biological systems. Fabrication details and sensor characterization are used to demonstrate the potential of these sensor concepts.

We report results of a study using differential phase optical coherence tomography (DP-OCT) for measurement of variation of refractive index (n) vs. analyte concentration (C) in translucent solutions and turbid tissue phantoms. Variation of refractive index with analyte concentration (dn/dC) in aqueous solutions of glucose, calcium chloride, magnesium chloride, sodium chloride, potassium chloride, potassium bicarbonate, urea, bovine serum albumin, and bovine globulin was measured. Obtained results demonstrated: (1) dn/dC for glucose is significantly greater than that of other analytes in the physiological range; and (2) high sensitivity of DP-OCT method for measurement of analyte concentration.

We study the consequences of the laser speckle phenomenon for the quantitative properties of laser Doppler perfusion imaging (LDPI) systems. These consequences are suggested by our earlier presented theory that allows for the prediction of the power of the photocurrent fluctuations in laser Doppler blood flowmetry (LDF). The speckle behaviour is particularly important in LDPI since tissue is illuminated by a free laser beam. In that case, the optical properties of the tissue govern the size of the speckles, together with the beam size and shape. Hence, the type of tissue affects the instrumental response through the speckles in an independent way. In this paper, this effect will be demonstrated experimentally, using diluted suspensions of Intralipid. We demonstrate that the photocurrent fluctuations decrease with increasing beam size and decreasing scattering coefficient of the Intralipid. The dependency on scattering becomes smaller with increasing beam size. Furthermore, we show that the power spectrum, which represents the Doppler shifts, becomes more narrow when the beam size is reduced. This can be explained by the relatively smaller speckle size associated with light that a longer path through the medium.

A sensor has been developed that uses multiple source excitation to measure blood perfusion in transplanted organs. To better isolate the signal of interest, wavelet decomposition analysis was used and compared to Fast Fourier Transform analysis. Data was collected in vitro using an adjustable peristaltic perfusion system and compared to simulated data created using low frequency sine waves. Standard FFT analysis and wavelet decomposition, using the symlet-4 wavelet mother function, was performed on both sets of data. The results showed that wavelet analysis was more suitable than FFT to extract the semi-periodic perfusion signal. These results indicate the potential of wavelet analysis for blood perfusion monitoring.

The deficiencies of the currently used pulse oximeter are discussed in diverse literature. A hazardous pitfalls of this method is that the pulse oximeter will not detect carboxyhemoglobin (COHb) and methemoglobin (metHb) concentrations. This leads to incorrect measurement of oxygen saturation by carbon monoxide poisoning and methemoglobinemia. Also the total hemoglobin concentration will not be considered and can only be measured in-vitro up to now. A second pitfall of the standard pulse oximetry is that it will not be able to show a result by low perfusion of tissues. This case is available inter alia when the patient is under shock or has a low blood pressure. The new non-invasive system we designed measures the actual (fractional) oxygen saturation and hemoglobin concentration. It will enable us also to measure COHb and metHb. The measurement can be applied at better perfused body central parts. Four or more light emitting diodes (LEDs) or laser diodes (LDs) and five photodiodes (PDs) are used. The reflected light signal detected by photodiodes is processed using a modified Lambert-Beer law (I=I₀•e^-α.d ). According to this law, when a non scattering probe is irradiated with light having the incident intensity I0, the intensity of transmitted light I decays exponentially with the absorption coefficient a of that probe and its thickness d. Modifications of this law have been performed following the theoretical developed models in literature, Monte Carlo simulation and experimental measurement.

A Multi-Layer Monte Carlo (MLMC) model was developed to predict the results of in vivo blood perfusion and oxygenation measurement of transplanted organs as measured by an indwelling optical sensor. A sensor has been developed which uses three-source excitation in the red and infrared ranges (660, 810, 940 nm). In vitro data was taken using this sensor by changing the oxygenation state of whole blood and passing it through a single-tube pump system wrapped in bovine liver tissue. The collected data showed that the red signal increased as blood oxygenation increased and infrared signal decreased. The center wavelength of 810 nanometers was shown to be quite indifferent to blood oxygenation change. A model was developed using MLMC code that sampled the wavelength range from 600-1000 nanometers every 6 nanometers. Using scattering and absorption data for blood and liver tissue within this wavelength range, a five-layer model was developed (tissue, clear tubing, blood, clear tubing, tissue). The theoretical data generated from this model was compared to the in vitro data and showed good correlation with changing blood oxygenation.

Velocity fields of blood flow in a micro channel were investigated experimentally using a micro-PIV velocity field measurement technique. The results were compared with those obtained for DI water under the same experimental solution. Diluted blood flow shows substantial variation of velocity in the central region of a micro-channel due to the presence of red blood cells, compared with DI water.

Fourier transform Raman and IR spectra are examined for malignant and normal breast tissues. Aside from individual differences that result in normal variations in lipids and glycogen, a clear distinction between normal and malignant tissues can be observed. MIR absorption spectra was observed using FTIR-Raman Spectrometer technique working in the regions Mir, Nir, Fir as well as VIS. Also the system can record Raman Scattering in the region from -2000 to 5600 cm-1 using Nd-Yag laser source of power 1500 mW. The comparison of Mid FTIR spectra from different samples of breast tumors with normal spectra reveals very important features that can be usefulk as diagnostic techniques. An absorption line centered at 3303 cm^-1 varies considerably and depends on the nature of the lesion, lipids and proteins composition. The ratio (I₃₄₄₃/I₄₃₅₂) has an average value of 2.4±0.3 for normal tissues while in the case of malignant tissues its average is 4.5±2.8. A broad absorption band was observed in some malignant cases between 2800-3750 cm^-1. A good shaped lines at 4259 and 4332 cm^-1 is clear in normal spectra, these lines tend to disappear in malignant cases. A normally existing line at 3470 cm^-1 increases considerably in malignant cases. When the tumor is not yet malignant it seems that the lines at 4259 and 4320 cm^-1 disappears first rapidly. As the tumor turns to be malignant, the band at 3470 cm^-1 increases and a new well defined band at 5167 cm^-1 appears. Moreover, Raman spectra showed the appearance of a new band at 2900 cm^-1 in the case of malignancy. While in normal breast samples this band didn't show up. Datat obtained in the NIR region confirms our observation in the MIR region.

Raman spectroscopy is a powerful technique as it provides fundamental information about vibrational modes of a system. Eigenvalues of these modes are very sensitive to the strength of the chemical bonds and perturbations caused by the environment, particularly charge distribution and alterations in the dipole strength of the system. All these parameters are profoundly modified during the tumor formation process nad hence Raman technique could be a unique and also a direct approach for evaluating tumor genesis at early stages. for this pupose the present investigation was carried out. We used cultured wild type and c-ras transformed NIH 3T3 fibroblast. The samples were treated with methyl alcohol solution ina conventional manner and then Raman spectra nad images were obtained by a specially developed confocal Raman microscope. The present results reveal differences between both cell types in the spectral details as well as in the morphology. Raman images are able to detect the exact site where cancer-related changes have taken place. These results clearly indicate the superiority of the present technique over conventional methods such as images obtained by X-rays or Nuclear Magnetic Resonance (NMR). Moreover, unlike other approaches, Raman images detect alterations at the submicron level rather than in the centimeter or millimeter range. Being an optical method it can be applied in vivo as a non-invasive technique potentially useful to early detection of cancer (particularly easy accessible cancers such as those of the skin and the digestive tract). The obtained resulsts suggest the great potential of Raman imaging in premature clinical diagnostic approaches.

We report on the scattering properties of oxygenated and de-oxygenated whole blood from 250-1000 nm. We determined the complex refractive index of oxygenated and de-oxygenated hemoglobin using Kramers Kronig analysis and Optical Coherence Tomography measurements. Combining these data with Mie theory, the scattering properties of oxygenated and deoxygenated whole blood were calculated. The results show strong oxygen saturation dependent scattering effects, which should be taken into account in data analysis of optical oxymetry.

We describe a non-invasive in vivo hyperspectral imaging technique for visualizing the spatial distribution of retina and optic nerve head (ONH) tissue oxygenation. Real time images of the fundus are acquired with continuous wavelengths (410-918 nm) to generate a data cube consisting of one spectral and two spatial dimensions. Reflected light from the one-dimensional (1-D) area of the sample is first passed through a grating and is then imaged onto a 12-bit silicone charge- coupled device (CCD) detector. A scanner then proceeds to the next 1-D area of the sample. Acquired image frames contain 256 spatial pixels and 256 wavelengths along rows and columns. Image sequences are scanned along the perpendicular spatial dimension using the push-broom method, whereby the spectrograph and camera are translated under constant velocity with respect to the fundus camera image over 6.6 mm of travel. This set of acquired images contains the full reflected light spectrum at each pixel of a two dimensional area of the retina and ONH. The system employs a focal plane scanner (FPS) using a linear actuator to provide motion. An algorithm processes spectral information at each pixel to represent the varying spatial distribution of retina and ONH tissue oxygenation. Imaging data are obtained from ONH tissue at both normal intraocular pressure (IOP) and acutely raised IOP.

The remodeling of cardiac tissues has been implicated in the development of congestive heart failure. Therefore, the current emphasis in cardiovascular research is geared toward understanding the underlying processes in order to facilitate the development of better prevention and treatment regimens by improving the early detection and diagnosis of this disease. This paper focuses on the application of polarized light to address a major drawback of cardiovascular biomechanics research, which is the utilization of toxic chemicals to prepare samples for histological examination. To accomplish this without the use of toxic chemicals, a polarization microscopy imaging technique was developed and applied to a non-stained rat septum sample. This imaging technique provided sufficient enhancement of collagenous structures to determine the myo-lamina sheet angle, β, without the need for caustic staining.

Current mammalian bioreporters using either firefly luciferase (luc) or GFP constructs require lysis and/or exogenous excitation to evoke a measurable response. Consequently, these cells cannot serve as continuous, on-line monitoring devices for in vivo imaging. Bacterial luciferase, lux, produces a photonic reaction that is cyclic, resulting in autonomous signal generation without the requirement for exogenous substrates or external activation. Therefore, lux-based bioluminescent bioreporters are the only truly autonomous light-generating sensors in existence. Unfortunately, the bacterial lux system has not yet been efficiently expressed in mammalian cells. In this research, three approaches for optimal expression of the a and b subunits of the bacterial luciferase protein were compared and reporter signal stability was evaluated from stably transfected human embryonic kidney cells. Maximum light levels were obtained from cells expressing the luciferase subunits linked with an internal ribosomal entry site (IRES). Cells harboring this construct produced bioluminescence equaling 2.6 X 106 photons/sec compared to 7.2 X 104 photons/sec obtained from cells expressing the luciferase from a dual promoter vector and 3.5 X 104 photons/sec from a Lux fusion protein. Furthermore, the bioluminescence levels remained stable for more than forty cell passages (5 months) in the absence of antibiotic selection. After this time, bioluminescence signals dropped at a rate of approximately 5% per cell passage. These data indicate that mammalian cell lines can be engineered to efficiently express the bacterial lux system, thus lending themselves to possible long-term continuous monitoring or imaging applications in vivo.

An integrated cytometric fluorescent imaging system is developed for characterizing chemical concentration and cellular status in microscale cell culture analog (μCCA) devices. A μCCA is used to evaluate the potential toxicity and efficacy of proposed pharmaceutical treatment of animals or humans. The imaging system, based on discrete optical components, not only provides a robust and compact tool for real-time measurements, but the modularity of the system also offers flexibility to be applicable to various μCCA structures that may be appropriate to various animal or human models. We investigate the dynamics of doxorubicin, a chemotherapeutic agent, on cultured cells in a μCCA using the integrated cytometric fluorescent imaging system. This study incorporates two uteran cancer cell lines representing a sensitive cell type and a multi-drug resistant (MDR) derivative cell line. The ultimate goal is to test the effect of MDR modulators in combination with doxorubicin to kill cancer cells while not causing undue harm to normal cells.

The x-ray PIV technique was empoyed to measure instantaneous velocity fields of blood flow in an opaque microchannel. Generally, it is difficult for conventional clinical instruments to visualize detailed transport of blood cells or to acquire quantitative hemodynamic information due to poor spatial resolution. On the other hand, although conventional PIV techniques have higher spatial resolution, they can be applicable only to transparent fluids inside a clear conduit. In addition, seeding particles indespensable for PIV measurements can affect the biochemical characteristics of blood. In order to resolve these problems, we established an x-ray PIV technique by using a permeable x-ray beam as a light source of PIV technique. For acquiring good x-ray images of red blood cells from which velocity vectors can be extracted, the sample-detector distance and the thickness of sample fluid were optimized. By applying 2-frame PIV algorithm to the acquired x-ray images of redd blood cells, the quantitative velocity field informatin was obtained. The measured velocity data of blood flow show typical flow characteristics in a macrochannel.

The photoacoustic technique is based on the absorption of modulated light by a sample and subsequent heat generation. This generates thermal waves that propagate in the surrounding media. According to the Rosencwaig-Gersho Model, such waves produce the pressure oscillation detected as the photoacoustic signal. This technique allows the spectroscopic characterization of multilayer systems: as the thermal diffusion length varies with the modulation frequency of the absorbed light, the depth profile of a sample can be studied by the analysis of the photoacoustic signal at different modulation frequencies. In this work, photoacoustic spectroscopy was used to characterize different human skin samples. Measurements were performed at 70Hz and 17Hz, using a 1000W Xe arc lamp as the light source, for wavelengths between 240nm and 700nm. Skin samples were about 0,5cm diameter. It was possible to obtain the photoacoustic absorption spectra of the stratum corneum and of a deeper layer of epidermis; when the lower modulation frequency is utilized, photoacoustic spectroscopy characterizes the absorption of the whole epidermis, because in this case the thermal diffusion length is thicker than that of the stratum corneum. Photoacoustic spectroscopy was also employed to monitor the drying kinetics of the skin. This was done by analyzing the time evolution of the photoacoustic spectra of skin samples. Pre-treatment of the samples included different periods in a drying chamber. Measurements show that the photoacoustic spectra changes according to the humidity of the skin. Future work includes detailed monitoring of skin hydration.

In the photoacoustic technique, the signal is proportional to the heat produced in a sample as a consequence of modulated light absorption. This technique allows the spectroscopic characterization of multilayer systems: as the thermal diffusion length varies with the light modulation frequency, one can obtain the depth profile of the sample by analyzing the frequency-dependence of the signal. As the photoacoustic signal depends on thermal and optical properties of the sample, structural changes in the system under analysis account for signal variations in time. In this work, photoacoustic spectroscopy was used to characterize samples of sunscreen and the system formed by sunscreen plus skin. We used photoacoustic spectroscopy to monitor the absorption kinetics of sunscreen applied to samples of human skin, characterizing alterations in the human skin after application of sunscreen. Measurements used 250W Xe arc lamp as light source, for wavelengths between 240nm and 400nm. This range corresponds to most of the UV radiation that reaches Earth. Skin samples were about 0,5cm diameter. The absorption spectra of sunscreen was obtained. Finally, photoacoustics was employed to monitor the absorption kinetics of the sunscreen applied to skin samples. This was done by applying sunscreen in a skin sample and recording the photoacoustic spectra in regular time intervals, up to 90 minutes after application. According to measurements, light absorption by the system sunscreen plus skin stabilizes between 25 and 45 minutes after sunscreen application. Results show that this technique can be utilized to monitor drug delivery and farmacokinetics in skin samples.

Capillaries play a critical role in cardiovascular function as the point of exchange of nutrients and waste products between tissues and circulation. A common problem for healthy volunteers examined during isolation, and for the patients suffering from heart failure is a quantitative estimation tissue oedema. Until now, objective assessment body fluids retention in tissues did not exist. Optical imaging of living capillaries is a challenging and medically important scientific problem. Goal of the investigation was to study dynamic of microcriculation parameters including tissue oedema in healthy volunteers during extended isolation and relative hypokinesia as a model of mission to the International Space Station. The other aim was to study dynamic of microcirculation parameters including tissue oedema in patients suffering from heart failure under treatment. Healthy volunteers and patients. We studied four healthy male subjects at the age of 41, 37, 40, and 48 before the experiment (June 1999), and during the 240-d isolation period starting from July3, 1999. Unique hermetic chambers with artidicial environmental parameters allowed performing this study with maximum similarity to real conditions in the International Space Station (ISS). With the regularity of 3 times a week at the same time, each subject recorded three video episodes with the total length of one-minute using the optical computerized capillaroscope for noninvasive measurement of the capillary diameters sizes, capillary blood velocity as well as the size of the perivascular zone. All this parameters of microcirculation determined during three weeks in 15 patients (10 male, 5 female, aged 62,2±8,8) suffering from heart failure under Furosemid 40 mg 2 times a week, as diuretic. Results. About 1500 episodes recorded on laser disks and analyzed during this experiment. Every subject had wave-like variations of capillary blood velocity within the minute, week, and month ranges. It was found that the perivascular zone sizes rising during isolation correlate with body mass of subjects and probably depend on retention of body fluids in tissues. Computerized capillaroscopy provides a new opportunity for non-invasive quantitative estimation tissue oedema and suggests for exact management patients suffering from heart failure under diuretic treatment.

It has been established that varicose veins are better visualized with infrared photography. As near-infrared films are nowadays hard to get and to develop in the digital world, we investigated the use of digital photography of varicose veins. Topics that are discussed are illumination setup, photography and digital image enhancement and analysis.

Peculiarities of light transport in Intralipid^TM solutions and the effect of glucose on light scattering properties of the solution at two different Intralipid^TM concentrations were studied with optical coherence tomography (OCT) technique in vitro. An open air OCT system using a superluminescent light source with center wavelength = 830 nm was used. 5% Intralipid^TM solutions were used to simulate a biological tissue (skin) in our experiment. Glucose concentrations at the physiologically relevant level were added to Intralipid^TM solutions. Increasing Intralipid^TM concentration increases the scattering coefficient of the media meanwhile increasing glucose concentration increases the refractive index of the media and reduces the scattering coefficient of the media. The experimental data were compared to Monte Carlo simulations. We also made the simulations for 2% Intralipid^TM solution. The results indicate that glucose added to 2 and 5% Intralipid^TM solutions changes their scattering properties, which is manifested by a decrease in the slope of the OCT signal. This finding shows the ways of using OCT for sensing glucose and monitoring the alterations of its content in biotissues. Some discrepancies between measurements and simulations were found, which need further investigation.

Integrins play an important role in the adhesion of cells to extracellular matrix and to other cells around them, more specifically fibronectin. The ultimate goal of this research is to detect these integrins on the surface of the cell with a combined atomic force microscopy (AFM) system coupled with a surface enhanced Raman spectroscopy (SERS) system. For this paper the focus was on identifying whether SERS is capable of being used to generate a unique spectrum for integrins. This was done using silver colloidal particles and the integrins a5B1 and aVB3. It was shown that a unique spectrum could be identified for each of these integrins at the nanomolar level.

We present the technical integration of state-of-the-art picosecond diode laser sources and data acquisition electronics in conventional laser scanning microscopes. This offers users of laser scanning microscopes an easy upgrade path towards time-resolved measurements. Our setup uses picosecond diode lasers from 375 nm, 405 nm, 440 nm and 470 nm for fluorescence excitation which are coupled in through a sole single mode fiber. The detected signal is guided to a photon counting detector, such as Photomultiplier Tubes (PMT) or Single Photon Avalanche Diodes (SPAD). This combines the outstanding sensitivity of photon counting detectors with the ease of use of diode laser sources, to allow time-resolved measurements of fluorescence decays with resolutions down to picoseconds. The synchronization signals from the laser scanning microscope are fed into the data stream recorded by the TimeHarp 200 TCSPC5,7 system, via the unique Time-Tagged Time-Resolved (TTTR)6 data acquisition mode. In this TTTR data acquisition mode each photon is recorded individually with its specific parameters as detector channel, picosecond timing, global arrival time and, in this special application, up to three additional markers. These markers, in combination with the global arrival time, allow the system software to reconstruct the complete image and subsequently fit the full fluorescence lifetime image. The multi-parameter data acquisition scheme of the TimeHarp 200 electronics not only records each parameter individually, but offers in addition the opportunity to analyse the parameter dependencies in a multitude of different ways. This method allows not only to calculate the fluorescence fluctuation correlation function (FCS) on any single spot of interest but also to reconstruct the fluorescence decay of each image pixel and detector channel for the purpose of Fluorescence Lifetime Imaging (FLIM) or advanced Fluorescence Resonance Energy Transfer (FRET) analysis. We present here some selected results acquired with standard laser scanning microscopes upgraded for the time-correlated single photon counting technique.