Non-invasive blood glucose monitoring has long been proposed as a means for advancing the management of diabetes through increased measurement and control. The use of a near-infrared, NIR, spectroscopy based methodology for noninvasive monitoring has been pursued by a number of groups. The accuracy of the NIR measurement technology is limited by challenges related to the instrumentation, the heterogeneity and time-variant nature of skin tissue, and the complexity of the calibration methodology. In this work, we discuss results from a clinical study that targeted the evaluation of individual calibrations for each subject based on a series of controlled calibration visits. While the customization of the calibrations to individuals was intended to reduce model complexity, the extensive requirements for each individual set of calibration data were difficult to achieve and required several days of measurement. Through the careful selection of a small subset of data from all samples collected on the 138 study participants in a previous study, we have developed a methodology for applying a single standard calibration to multiple persons. The standard calibrations have been applied to a plurality of individuals and shown to be persistent over periods greater than 24 weeks.

The ability of Kromoscopy to measure glucose selectively is demonstrated in solutions composed glucose, urea, triacetin, bovine serum albumin (BSA), cholesterol, and hemoglobin (Hb). Kromoscopic measurements are made with a four-channel instrument designed for measuring light between 1500 and 1900 nm. The channels are configured to respond to four individual bands of near infrared light centered at 1600, 1700, 1750, and 1800 nm. An equation is proposed that describes the relative response for each channel as a function of relevant experimental parameters. This equation predicts the linear response observed for these types of measurements as a function of solute concentration. In addition, molar absorptivities are provided for glucose, urea, triacetin, BSA, Hb, and water. The non-negligible absorptivity of water demands the consideration of water displacement caused by solute dissolution. Channel responses are measured for a series of thirty-one samples. The chemical composition of these samples is designed to minimize the correlations between glucose concentration and the concentrations of all other solutes. Likewise, these samples provide negligible correlation between the concentration of glucose and the extent of water displacement. A calibration model is constructed for glucose by using a conventional P-matrix multiple linear regression analysis of the four-channel information. The resulting model demonstrates selectivity for glucose with values of 1.27 and 1.34 mM for the standard errors of calibration and prediction, respectively, over a glucose concentration range of 1.9 to 19 mM.

For monitoring the blood glucose level noninvasively from the skin spectra, improvement of signal to noise ratio (S/N) of the glucose signal is critical. This cannot be achieved by the reduction of instrumental noise alone. To reduce the interference from undesired optical signals arising from the stratum corneum and the subcutaneous tissue, we designed a novel optical fiber probe for the skin spectra. The probe consisted of one central optical fiber around which several optical fibers were arranged in circle. The separation of the central optical fiber from each of the surrounding fibers was set at less than 1 mm. This probe was attached to the skin surface vertically when spectral measurements were performed. The measuring light was shone onto the skin surface through the circle fibers and scattered light reaching the central detecting fiber was collected and transmitted to the detection system. The true light path is not defined at present, but light passing through a long path could be neglected with this geometry. When we choose an adequate fiber distance, we can measure the dermis spectra selectively. Glucose intake experiments were performed with volunteers, for whom near-infrared (NIR) spectra were measured at the forearm, from which the blood glucose level was calculated. Partial least square regression (PLSR) analysis was carried out and we found good correlation between the optically estimated values of the glucose level and directly measured values of blood samples. The correlation coefficient characteristic had a positive peak at around 1600 nm, a typical of the glucose spectrum. In conclusion, our system using the novel optical fiber probe detected the changes in the glucose in the human skin tissue quantitatively and noninvasively.

We have analyzed the light propagation in tissue simulating media by a Monte Carlo method, using the estimated changes in the optical properties caused by variation of glucose concentration and temperature of the media. Using the calculated data, we have predicted the changes in the absorbance spectra caused by the change in glucose concentration in the absorbing and scattering medium. The predicted spectra have agreed very well with those obtained by the experiments. We have also studied the effects of the varying scattering coefficient on the spectra, and found that the changes in the absolute value and the wavelength dependency of the scattering coefficient are closely related to the changes in the observed spectra. In addition, we have calculated the changes in the absorbance spectra, when the glucose concentration and the temperature of glucose solution vary simultaneously. Using the multivariate analysis, we have extracted the glucose concentration accurately from the calculated absorbance spectra.

Noninvasive monitoring of analytes can be performed with optical coherence tomography (OCT) technique. This technique may allow measurement of optical properties of tissue (attenuation, scattering, optical thickness, etc.) that may be dependent on analyte concentration. Accurate monitoring of analyte concentration requires measurement of the optical properties with high accuracy. The accuracy of measurements depends on OCT technical characteristics and the level of speckle noise. In this paper, we report the results of the calibration of OCT system sensitivity for absolute and relative measurements of the backscattering and total attenuation coefficients in scattering standard, tissue phantoms (suspensions of polystyrene microspheres in water solutions of glucose), and human skin. We measured the OCT sensitivity as a function of depth and used this dependence for correction of signals. The amplitude and spatial period of backscattered signal modulation resulted from speckle noise were measured for the scattering standard and human skin. The dependence of speckle and electronic noise on the range of spatial and temporal averaging of OCT signals was determined. Our studies show that the accuracy of measurement of changes in optical properties of tissue with OCT technique can be significantly improved by reducing of speckle noise and by using the signal correction algorithm.

Self-assembled thin films containing embedded enzymes and fluorescent indicators are being developed for use as highly specific glucose biosensors. The sensors are fabricated using electrostatic Layer-by-Layer (LBL) adsorption to create oxygen-sensitive (Ruthenium-based) layers, the fluorescent intensity of which responds to changes in local oxygen levels. Oxygen is consumed locally by the reaction between glucose oxidase (GOx) molecules and glucose. Latex particles serve as the templates for our sensors and fabrication is carried out through the alternate adsorption of multiple levels of {GOx/polycation} and {Ruthenium-polycation/polyanion} bilayers. Additional fluorescence layers as well as fluorescent latex are being considered as internal intensity references to allow ratiometric monitoring. Films adsorbed to the nanoparticle templates are being studied to understand the fundamental chemical and optical properties, including enzymatic activity, spectral shape and emission intensity. Enzymatic activity is retained and stability is improved after adsorption, and increased surface area afforded by the particles allows use of increased numbers of molecules. Fluorescence is also maintained, though blue shifts are observed in emission spectra, and indicator activity remains. In vitro characterization studies demonstrate the feasibility of the particles as glucose biosensors, and future work will aim to optimize the response for neural monitoring.

Glucose monitoring is of critical importance in the life of Type I and many Type II diabetics. This research furthers work toward a minimally invasive implantable glucose sensor based on fluorescence detection. Current experimental models use heterogeneous fluorescence resonance energy transfer (FRET) systems for sensing; ideally, the response of one fluorophore bound to a large polysaccharide is enhanced greatly in the presence of glucose while the other fluorophore bound to a glucose sensitive protein is diminished or unaffected. Many fluorophores are affected by environmental factors such as pH and temperature. FRET experiments using two fluorophores, tetramethylrodamine isothiocyanate (TRITC) and fluoroscein isothiocyanate (FITC), are performed evaluating the effects of fluctuations over the range of pH 4-8 and temperature 25-45 degree(s)C for various concentrations of glucose in a flow cell. TRITC is bound to the lectin Concanavalin A (Con A), and FITC is bound to dextran molecules of varying sizes.

Hollow microshells are being fabricated for potential use as versatile sensors for real-time measurements of biochemicals. These shells are assembled using Layer-by-Layer (LBL) assembly of polyions onto colloidal polymer template particles. The latex particles are subsequently dissolved leaving behind stable, hollow shells into which analyte-sensitive and reference dyes are introduced. Fundamental studies have been performed to determine optical and chemical characteristics of these shells. Tests have been performed to determine the integrity of shells with respect to leaching and structural stability and robustness. The microshells were then loaded with an assay composed of a sodium sensitive fluorophore (SBFI) or a potassium sensitive fluorophore (PBFI), along with an analyte insensitive reference dye. It has been shown that the dye-loaded capsules retain their sensitivity to ion concentration with fluorescence characteristics similar to those of the dyes in liquid-phase, and that the sensor response to increasing ion concentration is linear over a physiologically significant range. In addition, preliminary results have demonstrated the ability of these capsules to be loaded with other sensor chemistry, including Ruthenium/Glucose-oxidase, and a FITC-dextran/TRITC-ConA competitive binding assay for glucose sensing based on fluorescent resonance energy transfer (FRET) methods.

Much work has been done to make the optical measurement of glucose concentrations in the aqueous humor a feasible, non- invasive, alternative for the blood finger stick, method currently used by people with diabetes mellitus. Recent work has demonstrated that the time lag between blood and aqueous humor glucose levels is within five minutes but there is still work to be done in overcoming the confounding effects of changing birefringence, due to motion artifact, on the detected glucose signal during in-vivo measurements. To address this issue, we designed and implemented a dual orthogonal polarization detection system. We present promising preliminary results that indicate that this method, with some slight modifications and optimization of our system, has the potential to extract glucose concentration information from a birefringent sample in the presence of motion artifact.

Low molecular weight molecules are typically very difficult to detect directly in solution using commercially available SPR (surface plasmon resonance) instruments. This is because the mass change on binding is not sufficient to cause a detectable change in refractive index on binding to surface-bound receptors (e.g., antibodies). Some receptors, however, undergo extensive changes in tertiary structure upon binding ligands. Here we present data suggesting conformational changes in surface-bound receptors such as periplasmic binding proteins and calcium-binding proteins can be detected by SPR. This SPR response can be used to monitor specific binding of carbohydrates and calcium even though the molecular weight of these analytes would be difficult to detect using traditional SPR methods. Therefore this approach has potential applications for developing optical biosensors for such small molecules.

Low molecular weight molecules are typically very difficult to detect directly in solution using commercially available SPR (surface plasmon resonance) instruments. This is because the mass change on binding is not sufficient to cause a detectable change in refractive index on binding to surface- bound receptors (e.g., antibodies). Some receptors, however, undergo extensive changes in tertiary structure upon binding ligands. Here we present data suggesting conformational changes in surface-bound receptors such as periplasmic binding proteins and calcium-binding proteins can be detected by SPR. This SPR response can be used to monitor specific binding of carbohydrates and calcium even though the molecular weight of these analytes would be difficult to detect using traditional SPR methods. Therefore this approach has potential applications for developing optical biosensors for such small molecules.

We describe on-line optical measurements of urea concentration during the regular hemodialysis treatment of several patients. The spectral measurements were performed in the effluent dialysate stream after the dialysis membrane using an FTIR spectrometer equipped with a flow-through cell. Spectra were recorded across the 5000-4000 cm^-1 (2.0-2.5 micrometers at 1-minute intervals. Optically determined concentrations matched concentrations obtained from standard chemical assays with a root-mean-square error of 0.29 mM for urea (0.8 mg/dl urea nitrogen), 0.03 mM for creatinine, 0.11 mM for lactate, and 0.22 mM for glucose. The observed concentration ranges were 0-11 mM for urea, 0-0.35 mM for creatinine, 0-0.75 mM for lactate, and 9-12.5 mM for glucose.

The microvascular perfusion can be measured using laser Doppler blood flowmetry (LDF), a technique sensitive to the concentration of moving blood cells and their velocity. However, movements of the tissue itself can cause artifacts in the perfusion readings. In a clinical situation, these movement induced artifacts may arise from patient movements or from movements of internal organs e.g. the intestines or the beating heart. Therefore, we have studied how a well-controlled tissue movement affects the LDF signals during different flow conditions and for different surface structures. Tissue perfusion was recorded non-touch in one point using a laser Doppler perfusion imager. During the measurements the object was placed on a shaker that generated the movement (both horizontal and vertical). Measurements were carried out both on DELRIN (polyacetal plastic) and the fingertip, for a wide range of velocities (0-3 cm/s). The influence of the microvascular perfusion was evaluated by occluding the brachial artery as well as blood emptying the finger and by using a flow model. The LDF signals were correlated to the movement. In vivo measurements showed that velocities above 0.8 cm/s gave a significant contribution to the perfusion signal. Corresponding velocities for the DELRIN piece were higher (1.4 - 2.6 cm/s), and dependent on the surface structures and reflecting properties. By reducing the amount of specular reflection the movement influence was substantially lowered.

Goals of the investigation were to analyze the spectrum of microcirculation parameters and collection of baseline data healthy subjects during extended isolation and relative hypokinesia as a model of mission to the International space station. There were investigated four healthy volunteers at the age of 37, 40, 41 and 48 during the baseline 240-d isolation period starting from July 3, 1999. With the regularity of 3 times a week each subject made records at the same time between 1 and 2 pm. Optical computerized capillaroscope for noninvasive measurement of the capillary diameters, blood flow velocity as well as the size of the perivascular zone and the number of the blood aggregates was used. About 1500 episodes were recorded on laser disks and analyzed. Parameters of microcirculation were compared with other physiological parameters monitored in the experiment. All subjects had wave-like variations in the microcirculation parameters within the minute, week, and month ranges. Mean blood flow velocity in the baseline period was lower than in the period of isolation. Results of the daily body mass measurement were found to correlate with the perivascular zone size, that could be explained as retention of body fluids in tissues. Computerized capillaroscopy is easy to perform, noninvasive, highly sensitive and informative. It enables analysis of the character of rhythmic processes, adaptability of organism to long-term experiments and, therefore, can be proposed for use in extended space missions.

The experimental model was created during an abdominal dissection of the rat mesentery and ligation of the collecting vein of mesentery. It was found that mesentery edema was developed for 20-30 min. Simultaneously monitoring of main the parameters of blood and lymph flow revealed significant increasing of interstitial water amount (up to 3 times), reduction of blood microcirculation, increasing of blood vessels permeability, decreasing of lymph microvessels diameter, phasics contractions, and lymph flow velocity.

In this work we described the new modification of experimental setup designed on the basis of transmission microscopy and high-resolution speckle-correlation technique. This combined technique provides the simultaneous speckle and video registration of lymph dynamics that allows one to calibrate the speckle-correlation velocity sensor and to determine an absolute flow velocity and its direction. As a result many parameters of lymph dynamic were measured quickly, conveniently and simultaneously and a new data about the lymph flow velocity and other functions of microcirculation were received. The results of the experimental study of lymph microcirculation in small intestine mesentery of rat in vivo are presented.

The efficiency of the molecular machines can be determined by the Q-factor of oscillations in the selected degrees of freedom. In particular, the quasi-harmonic oscillations of chymotrypsin decrease the threshold for the diffusion limitation of its functioning as molecular scissors. It was demonstrated by the methods of molecular dynamics that the Q-factor of the subglobular oscillations of proteins in water at the frequencies (omega) about 10¹² Hz can reach 10, although the Raman bands in the corresponding spectral range were found only in crystal samples. Q quadratically increases with (omega) . The earlier hypothesis regarding the catalytic importance of the Fermi resonance for the oscillations of ligands and atomic groups in the enzyme- substrate complexes was not proved in experiments with simple models. The Fermi resonance can be observed if the Q- factor of the selected degree of freedom exceeds 100.

For the analysis of small concentrations of organics in aqueous solutions, a novel add-on accessory for dual- beam/optical subtraction spectroscopy has been built for a commercial Fourier transform infrared spectrometer. A standard FT-IR instrument requires a sample measurement and a separate reference measurement, whereas the optical subtraction instrument directly measures the difference between sample and reference. This has a number of advantages. The time delay between the two measurements is eliminated, and the effective measurement time is improved by a factor of two. Moreover, the optical subtraction provides a large reduction in dynamic range of the measured signal, which prevents detector saturation, and enables effective use of dynamic range of the analog to digital converter in the FT-IR spectrometer. This results in an increased signal to noise ratio, compared to the standard FT-IR instrument. By changing detector and light source the instrument may be used for both near- and mid-infrared spectroscopy. The increased sensitivity and stability of the optical subtraction instrument compared to the standard instrument is demonstrated by transmission measurements of aqueous urea solutions in the combination band region 4000 to 5999cm^-1(2000 to 2500 nm).

An important task of in vivo polarimetric glucose sensing is to find an appropriate way to optically access the aqueous humor of the human eye. In this paper two different approaches are analyzed theoretically and applied to the eye model of Le Grand. First approach is the tangential path of Cote et al., and the second is a new scheme of this paper of applying Brewster reflection off the eye lens.

We present a new detection instrument for chemical/biological fluorescence lifetime-based sensors. The instrument comprises a primary, closed loop with a secondary loop controlling a variable phase delay within the primary loop. The primary loop consists of a fluorescence excitation light source, a fiber-optic delay line (with a gap for placement of a fluorescent sensor), an electronic phase shifter, a photo-detector, and a resonance-type RF amplifier. The secondary loop consists of a long-wavelength-pass optical filter, multimode fiber, a PMT, and an electronic phase detector (which is connected to the phase shifter of the primary loop). The system exhibits self-oscillations in the form of RF sinusoidal intensity modulation with frequency dependent on the fluorescence lifetime. Since the primary loop does not contain an optical filter, it is easier to obtain self-oscillations (compared to single loop systems). The feedback also improves the stability of the detection platform. The detection system is simple, inexpensive, and scalable for sensor array purposes. We demonstrate the use of a cost-effective, multi-channel, computer-algorithm-based frequency counter with this new system. We illustrate the detection capabilities of this detection system with the pH-sensitive, fluorescent probe carboxy seminaphthofluorescein (SNAFL-2) and an immunosensor based on fluorescence resonance energy transfer.

Using angle-resolved Monte-Carlo simulation we obtained indicatrices of light scattering from a whole blood layer (suspension of erythrocytes at physiological hematocrit) for different wavelengths (514 and 633 nm) of incident light. We considered the problems of conformity of parameters describing the model medium and real investigated medium under experiment conditions (shape and size of particles, refractive indexes of particles and suspension, hematocrit). The anisotropy factor was numerically determined for various shapes of model erythrocytes: equivolumed spheres, chaotically oriented spheroids with axial ratio (epsilon) =0.25, and bi-concave disks. For calculations we used different approaches: the exact Mie theory for spheres; hybrid approximation, based on anomalous diffraction approach, for spheroids; and geometrical optics approximation for bi-concave disks and spheroids. The best fir for experimental results obtained at (lambda) =514 nm provided the calculations for the layer of erythrocytes, modeled by chaotically oriented spheroids, which phase functions were calculated by geometrical optics approximation (taking into account Franhofer diffraction). So we conclude that the average shape of erythrocyte in suspension is closer to the spheroid with axes ratio 0.25.