The selectivity of a four-channel Kromoscopic analysis is demonstrated for the measurement of glucose in separate binary and tertiary matrices. A novel virtual search procedure is used to identify three different sets of four, overlapping transmission filters. The first filter set includes filters centered at 900, 1300, 1410, and 1538 nm and is selected to differentiate glucose and urea in a series of binary mixtures. These binary mixtures were prepared with independent levels of 1-10 mM glucose and 9- 213 mM urea dissolved in an aqueous phosphate buffer solution. A second filter set contains filters centered at 1064, 1100, 1224, and 1290 nm and is used to measure glucose in a series of tertiary mixtures composed of glucose, urea and bovine serum albumin. This tertiary matrix consists of 2-13 mM glucose, 13-129 mM urea and 0.05-0.46 g/L bovine serum albumin dissolved in the same type of buffer. Multilinear regression is used to relate the relative Kromoscopic responses to the concentration of glucose in these sample solutions. In both cases, the prediction errors are on the order of 0.6-0.8 mM. The impact of solution temperature is also investigated by examining glucose responses obtained from solutions maintained at temperatures ranging from 35 to 39 degree(s)C. The filter set used in this experiment is composed of filters centered at 1100, 1150, 1254, and 1300 nm. Results from this particular filter set indicate that the directionality and magnitude of the glucose responses are independent of solution temperature. Finally, accurate glucose measurements are demonstrated when a same-temperature blank is used to generate the relative channel response.

Factors that have limited the acceptance of optical spectroscopy methods for non-invasive blood glucose sensing include signal variations due in part to changes in the skin tissue optics between patients, the lack of a repeatable pathlength inherent in using diffusely reflected photon approaches, temperature variations on the skin, and the pressure with which a probe is applied to the skin surface. Unfortunately, most previous approaches to non-invasive glucose sensing have failed to address these important issues. In this work, we developed a novel skin port sensor (SPS) which eliminates the effect of skin optics by using a stable, infection-free, dermal implant to provide a skinless window into the body. Our implant is designed to provide a fixed optical pathlength as well as features to minimize temperature and pressure variations. Preliminary experiments in a pig model demonstrate both a stable biological seal at the transcutaneous interface as well as ingrowth of vascular containing granulation tissue within the sensing chamber. Furthermore, optical spectra acquired from the port demonstrate changes in glucose signatures related to concentration changes induced in the blood. Our novel SPS may provide the necessary platform for successful implementation of an optical approach to in vivo glucose sensing.

Progress towards a painless and hygienic glucose monitoring procedure for diabetics continues as the growth of diabetes mellitus reaches epidemic proportions in the American population. Utilizing an implantable fluorescence based glucose assay, the minimally invasive approach presented here has previously shown promise towards this goal in terms of glucose specificity and quantification for in vitro environments. However, in realistic physiological circumstances the depth of the implant can vary and optical properties of skin can change due to normal physiological conditions. Additionally, naturally occurring auto-fluorescence can obscure the sensor signal. An important concern under these conditions is that variations of fluorescent intensity due to these or other causes might be mistaken for glucose concentration fluctuations. New data shows that fluorescence-based glucose assays can be probed and interpreted in terms of glucose concentrations through pig skin at depths of up to 700 mm when immobilized in a bio-compatible polymer. When a combination of two fluorophores are employed as demonstrated here, reasonable changes in skin thickness and the confounding effects of the variations inherent in skin can be overcome for this glucose sensing application.

The widespread occurrence of diabetes mellitus and the severity of its associated complications necessitate the development of non-invasive blood glucose measurement devices in an attempt to improve treatment regimens and curb the complications associated with this disease. One method showing promise in this endeavor utilizes optical polarimetry to monitor blood glucose levels indirectly by measuring glucose rotation of polarized light, which is a direct indication of glucose concentration, in the aqueous humor of the eye. The presence of other optically active (chiral) components in the aqueous humor of the eye have the potential to confound the glucose measurement of optical rotation when using a single wavelength polarimeter. Thus, this has led to the recent investigation of multispectral polarimetric systems which have the potential to enable the removal of confounder contributions to the net observed optical rotation, therefore, increasing glucose specificity and reducing glucose prediction errors. Such polarimetric systems take advantage of the uniqueness in the rotation of polarized light, as a function of wavelength, by the chiral molecule of interest. This is commonly referred to as the optical rotatory dispersion (ORD) spectra of the chiral molecule. ORD characterization of the chiral molecules within the aqueous humor is necessary for determining the optimum number of wavelengths needed to reduce glucose prediction errors; however, this information is often only given at the sodium-D line (589 nm) in the literature. This report describes the system we designed and built to measure ORD spectra for glucose and for albumin, the main optical confounder within the aqueous humor, as well as our investigation of the effects of temperature and pH on these ORD spectra.

Development of minimal invasive diagnostics methods is of great importance in today's healthcare. The present work deals with the measurements of glucose concentration in small drops, its volume being in the range of 1 (mu) L and its glucose concentration being in the range of 1-10 mM. The aim of the work was to develop robust quantitative method of glucose measurement in small volumes of interstitial fluid obtained after laser shot or by other low invasive procedures. The commercially available Amlex Red glucose assay kit was used in the present work. The glucose is detected by enzyme-coupling reactions resulting in formation of fluorescence dye resorufin. The diagnostic paper was used as a reaction medium. The fluorescence of dye in diagnostic paper was measured with the use of fiber optics spectrometer. It was shown that dye fluorescence correlates fairly well with glucose concentration. The method may be modified to involve cholesterol concentration measurement as well.

The aim of this work was to describe a new method for optical monitoring of solutes in a spent dialysate. The method utilizes UV light absorption employing a commercially available spectrophotometer. Measurements were performed both on collected dialysate samples and on-line. The concentration of several removed solutes and electrolytes in the serum and in the dialysate was determined simultaneously using standard laboratory techniques. During on-line monitoring the spectrophotometer was connected to the fluid outlet of the dialysis machine. On-line measurements during a single hemodialysis session demonstrated a possibility to monitor deviations in the dialysator performance (e.g. dialysator in by-pass). The experimental results indicated a good correlation between UV absorption and several removed solutes (urea, creatinine) in the spent dialysate. The correlation coefficient for urea and creatinine concentrations in the dialysate was very high for every individual treatment. The UV absorbance correlates well to the concentrations of several solutes thought to be uremic toxins. The results indicate that the technique can be used as a continuous, on-line method for monitoring deviations in the dialysator performance and may estimate the removal of the overall toxins. In the future, the new method will be used to evaluate parameters describing delivery of the prescribed treatment dose such as KT/V and Urea Reduction Rate (URR).

The authors report on the development of a novel technique for non-invasive measurements of the concentration of drugs in the anterior chamber of the eye. Presently there is no satisfactory, real-time method available to the ophthalmic community. Accurate determination of drug concentrations in the eye would be of great value and assistance to researchers and drug companies manufacturing ophthalmic drugs and ocular implants, enabling a better understanding of intra-ocular pharmacokinetics. At present researchers use techniques of direct sampling of the aqueous humour from laboratory animal eyes into which the drug has been introduced topically or systemically. Rabbit eyes are frequently used in this context. Sampling via paracentesis is invasive, can be painful and moreover does not yield a continuous measurement. Our approach to addressing this measurement requirement is, in effect to turn the eye into a cuvette and use optical absorbance spectroscopy measurements to obtain drug concentrations. A novel contact lens has been designed using commercial, off-the-shelf, optical design software. The lenses have been optimised to direct light across the anterior chamber of the rabbit eye. Practical demonstration and characterisation of light propagation across the eye has been undertaken and will be reported on. Preliminary results on the identification of drug compounds introduced into the animal and model eyes will be also reported.

There are correspondences between the number of lipid inclusions as detected with computerized capillaroscopy and the lipid parameters as determined using a routing blood test. The results have been obtained in the course of a study of two healthy volunteer groups (n equals 24) and three groups of patients: those suffering from ischemic heart disease with myocardial infarction in their past and heart failure (n equals 13), complicated insulin-independent diabetics (n equals 58), and patients with uncomplicated insulin-independent diabetes (n equals 27). Neither of the healthy volunteers had small lipid inclusions in the blood on an empty stomach. Moreover, lipid exchange parameters of every healthy volunteer as measured in a routine test were within the acceptable limits, while in patients with diabetes as well as in patients with coronary artery disease complicated by heart failure who had high cholesterol level, an elevated number of lipid inclusions was observed.

It was shown in our recent papers that distilled water possesses intrinsic luminescence at wavelength of about 400 nm with excitation wavelength 300 nm, which is very sensitive to small amount of dissolved substances. This phenomena was chosen to study homeopathic remedies. Pronounced difference in the intensity of luminescence between several commercial preparations with the same potency and one preparation with various potencies was obtained. Long scale evolution of the spectra was registered and final result was dependent on preparation and its potency. Systematic study of homeopathic preparations of halit (natural sodium chloride) from 1 to 30 decimal dilution was done. A stepwise dilution with mechanical agitation between the dilution steps, the so-called potentisation, was produced specially by homeopathic company Weleda. Luminescence intensity against concentration (potency) of halit is non monotonous function with several maxima, the main maximum is located at 13-14-th dilution. Evolution of the spectra was registered during several months. The analogous potentisation treatment of water without additional substances results also in changes of the luminescence spectra, depending on the number of potentisation. The obtained differences of luminescence spectra at ultra high dilutions and potentisation show that the collective properties of water are really changed in homeopathic preparations.

A compact laser Doppler flowmeter (35x80x210mm³) for the measurement of subfoveal choroidal blood flow parameters (ChBF) was mounted on a helmet. This device allows the measurement of ChBF during dynamic exercises or in supine position, without the need for pupil dilatation. Its optical system is based on a Schlieren arrangement by which the surface of light collection and that of the illumination are spatially separated by an obscuration. The laser probing beam ((lambda) equals 790 nm, 100 (mu) W at the cornea) is focused at the fovea by having the tested subject look directly at the beam. Computer analysis of the photocurrent produced by the scattered light provides a relative measure of the mean blood velocity, number and flux of the red blood cells in the choriocapillaris. Measurements were performed to assess the reliability of the flow parameters measurements in normal volunteers: reproducibility and sensibility when subjects are sitting or standing; measurement of changes in ChBF in the case of isometric and dynamic exercises. Results demonstrate that this new helmet-mounted device provides data comparable to the conventional device. It allows for the first time, however, the continuous measurement of choroidal hemodynamics in humans during various types of exercises.

In this paper an experimental test bench for mechanical heart valve and the procedure for non-invasive optical measurement are reported. Fluidynamic behaviour of a bileafleat mechanical valve in steady state and pulsed flow conditions has been studied. Laser Doppler Anemometry (LDA) is used to access velocity and turbulence values at different distances before and after the mechanical valve. Data obtained can be related, according to the literature, to typical pathologies affecting patients who underwent surgical procedures to implant mechanical heart valves. In particular thrombosis and hemolysis can be related to high levels of shear stress affecting blood cells. Measurements of velocity, turbulence and shear stresses have been performed.

Statistical analysis of images of time-integrated dynamic speckle patterns is considered as the tool for diagnostics and imaging of in vivo tissue dynamics such as blood microcirculation in superficial layers of human tissues and organs. Basic approach for blood microcirculation monitoring using the contrast analysis of time-averaged speckle images is known as LASCA (Laser Speckle Contrast Analysis) technique. This paper presents the modified version of LASCA, which is based on application of the localized probe light source and the spatial filtration of analyzed speckle pattern in the object plane. Being compared with classical LASCA technique, this method has the certain disadvantage as the necessity of scanning procedure to provide the reconstruction of maps of blood microcirculation parameters, but it gives the additional possibilities for the analysis of depth distributions of these parameters. Theoretical background for the depth-resolved analysis of blood microcirculation parameters on the basis of the concept of effective optical path distributions for multiply scattered probe light is considered. The effect of non-zero residual contrast even in the case of large integration times is also discussed.

The membranes of the erythrocytes are covered with different kinds of proteins determining decisively the aggregation and disaggregation properties of erythrocytes. Morbid changes in the human body are reflected by changes in the kind and concentration of these proteins and this way by the aggregation and disaggregation behavior of erythrocytes. Light scattering measurements were used for the investigation of these changes comparing healthy volunteers and patients with different diseases. The whole blood samples were analyzed using a rotating coaxial cylinder system in combination with a special method for data processing. The erythrocyte aggregates were destroyed completely without injuring the cell membranes by shear forces arising during the cylinder rotation. In this paper the results of aggregation and disaggregation analysis of erythrocytes in whole blood samples of healthy volunteers and patients suffering different diseases are presented. The method of erythrocyte aggregation and disaggregation measurements is compared with blood sedimentation measurements.

The ultimate purpose of our research is to demonstrate experimentally the relationship of the deformation and aggregation of red blood cells to the viscosity of blood. We need to measure simultaneously the viscosity of blood and the aggregation of red blood cells. Therefore, we propose a new method to measure simultaneously the viscosity of fluid and the aggregation of particles. The method is developed by combining a cone-plate viscometer with the technique of the particle sizing based on the dynamic light scattering. We show theoretically that a temporal autocorrelation function on the intensity basis is a square of a sum of the autocorrelation functions on the amplitude basis of light scattered from particles and the dynamic speckle produced from the rough surface of a rotating cone in the viscometer. The theoretical prediction is confirmed by experiments for using the solutions of polystyrene latex particles.

We prove experimentally that RBC aggregation is among the major factors affecting time evolution of light transmission in both the normal situation of pulsatile blood flow and the situation of over-systolic vessel occlusion. Optical transmissions of tissue in vivo have been measured in red/near-infrared region. Sudden blood flow cessation causes the light transmission rising. For certain wavelengths range this growth becomes non-monotonic. The correspondence between in vivo measurements and the theoretical simulations is reached if we attribute the transmission growth to the change of average size of scatterers. The most important blood parameters such as hemoglobin, glucose, oxygen saturation, etc., influence the transmission growth following over-systolic occlusion and, therefore, may be extracted from the detailed analysis of the time evolution of optical transmission. It forms a basis for new kind of non-invasive measurements, i.e., occlusion spectroscopy. The results of in vivo clinical trials are presented for glucose and hemoglobin.

We develop theoretical models of light transmission through whole blood considering RBC aggregation. RBC aggregates are considered to be the main centers of scattering in red/near- infrared spectral region. In pulsatile blood flow the periodic changes of aggregate geometry cause oscillations of light scattering. Thus scattering-assisted mechanism has to be taken into account in pulse oximeter calibration. In case of over-systolic vessel occlusion the size of aggregates grows, and the light transmission rises. Light diffraction on a single scatterer makes the transmission growth non- monotonic for certain spectral range. For the most typical set of aggregate parameters this range corresponds to wavelengths below 760 nm, and this prediction fits well both in vivo and in vitro experimental results. This spectral range depends on the refraction index mismatch and the geometry of aggregates. Both of them may be affected by the chemistry of blood. For instance, changes of glucose and hemoglobin have different effect on light transmission time response. Consequently, their content may be determined from time evolution of optical transmission.

The concept of refractive index matching used for the enhancement of optical penetration depth of the whole blood is discussed on the basis of in vitro studies using NIR optical coherence tomography technique. It was found that blood optical clearing is defined not only by refractive index matching effect, but also by changes of RBC size and their aggregation ability when chemicals are added. For example, for whole blood twice diluted by a saline adding of 6.5% of glycerol reduces the total attenuation coefficient from 4.2 mm^-1 to 2.0 mm^-1, and correspondingly increases the optical penetration at 820 nm up to 117%. For other tested agents (glucose, dextrans, propylene glycol, and trazograph) the enhancement of penetration was from about 20% up to 77%. In blood sedimentation study the regular or irregular oscillations or jumps of the RBC/plasma boundary were observed. The one- minute time period of regular oscillations well correlates with the kinetics of the aggregation process, described by the two subsequent stages of formation of the linear and 3D aggregates.

For the first time, the formation of macromolecular protein clusters in water solutions in the presence of heavy metal ions were observed by the light scattering method. Conditions of the formation and destruction of such clusters were investigated. The clusters mass has a maximum value at the isoelectric point of the protein and increases with the ionic strength of the solution. Formation of clusters in the presence of toxic heavy metals in the blood and living cells is believed to have an essential physiological meaning. The molecular mechanisms of clusters formation based on dipole- dipole interactions of the macromolecules are discussed.

The extinction of light passing through a blood vessel comprises both absorbed and scattered components, the latter of which includes relatively strong forwardly transmitted and directly reflected components. The effect of such vessels on incident light beams of arbitrary polarization is most thoroughly described by the vessel's transmission and reflection Mueller matrices. The Mueller matrices of illuminated mock blood vessels (diameter 102-278 micrometers ) in these two important directions have been measured at a wavelength of 633 nm using a Mueller matrix imaging polarimeter. The measured Mueller matrices are presented, decomposed, and analyzed to determine the sample's retardance and depolarization as a function of vessel diameter. It is expected that characterization of these matrices should broaden light-vessel modeling techniques by permitting calculation of the transmitted and reflected properties of arbitrary input polarization states.

We present a new approach to excited state fluorescence lifetime-based measurements which is inexpensive and highly sensitive. The detection system consists of a closed-loop optoelectronic arrangement containing an intermediate frequency resonance amplifier, a fluorescence excitation light source (for example, a light emitting diode or a semiconductor laser), a fiber optic delay line, and a photodetector. The system exhibits self-oscillations in the vicinity of the frequency (Omega) approximately 1/(tau) (where (tau) is the excited state lifetime) which manifest themselves as modulation of the light. Changes in the excited state lifetime alter the phase delay of the loop, which in turn causes a frequency shift in the modulation signal. The frequency shift can be measured very precisely with a frequency counter. With appropriate averaging, this technique can yield sub-picosecond resolution of shifts in lifetime. This technique is suited for chemical/biological sensing applications, and can be easily duplicated for chemical/biological sensor arrays.

Aiming at development of tools for accurate non-invasive diagnosis, we investigate the accuracy of experimental and numerical approaches for monitoring of deep structures, such as blood vessels, together with determination of their optical parameters. Among numerical techniques developed for determination of structure and optical parameters of biotissue basing on measurement data, Monte Carlo simulations is the best tool, but requires a considerable computation time to preserve accuracy when one deals with large source detector separations. In contrast, diffusion approximation is fast but suffers from a difficulty of estimation of accuracy. We implemented an intermediate approach, deriving and solving frequency-domain-specific equations basing on radiative transfer theory. We conducted measurements and corresponding simulations using multilayered tissue-simulating sample and estimate accuracy novel techniques can achieve. We obtained an accuracy of 0.002 mm^-1 in measurements of the reduced scattering coefficient.

The generation and propagation of fluorescence light through tissue offers the potential for biomedical diagnostics and clinical biosensing. Arising from an exogenous fluorescent dye injected as a contrast agent or immobilized in a polymer implant, the fluorescent decay kinetics can be sensitive to the biochemical environment of the tissue, providing quantitative in vivo information of the confined tissue site. To date, all studies of fluorescence in scattering media have been confined to dyes with single-exponential decay kinetics, yet most of sensing fluorophores exhibit multi-exponential decay. The goal of the study was to develop a time-dependent model describing the generation and propagation of fluorescent light emitted from dyes exhibiting multi-exponential decay kinetics. To experimentally investigate multi-exponential decays, we employed two dyes, 3,3-diethylthiatricarbocyanine iodide (DTTCI) and Indocynanine Green (ICG), which exhibit distinctly different lifetimes. The experimental study was divided into two parts. In the first part, fluorescence phase-modulation measurements were made as a function of modulation frequency at various concentration ratios of the two dyes in dilute nonscattering solutions. In the second part, frequency measurements of phase-modulation were made at similar concentration ratios as the first part, but in presence of scattering (2% intralipid). The multi-exponential decay parameters extracted from the phase-modulation data in the first part of the study were used to predict the phase-shift and amplitude-attenuation values in the presence of scattering. Using a diffusion model, the predictions were then compared to the second part of the experimental study.

Kinetics of photobleaching of background in Raman spectra of aqueous solutions of plant toxins ricin and ricin agglutinin, ricin binding subunit, and normal and malignant human blood serum were measured. For the excitation of the spectra cw and pulsed laser radiation were used. The spectra of Raman background change upon laser irradiation. Background intensity is lower for the samples with small molecular weight. The cyclization of amino acid residues in the toxin molecules as well as in human blood serum can be a reason of the Raman background. The model of the background photobleaching is proposed. The differences in photobleaching kinetics in the cases of cw and pulsed laser radiation are discussed. It is shown that Raman background photobleaching can be very informative for cancer diagnostics.

Combination of laser Doppler flowmetry and pulse oximetry methods allows for the direct assessment of oxygen supply to tissues at the microcirculatory level, namely, in that part of the vascular network where the transcapillary exchange takes place that is responsible for saturating tissues with oxygen. The microcirculation system comprises arterial and venous microvascular parts that differ in blood flow velocities. Frequency separation of the photodetector signal components related to different velocity ranges makes possible to distinguish the hemodynamic processes in these two parts of the microvascular system. Moreover, numerous studies of collective oscillatory processes in hemodynamics reveal that cardio-oscillations are more pronounced in arterioles, whereas venous hemodynamics is mostly influenced by the breath rhythm. Taking account of the above phenomena allows developing a signal-filtration system for separate characterization of blood-oxygenation states in arterial and venous blood flows. Light absorbance in the skin depends on both light wavelength and blood-oxygenation level. Processing the signals obtained with a two-channel dual-wavelength (630 and 1115 nm) laser Doppler flowmeter provides information about blood oxygenation levels at the entrance and exit of the microvascular system and allows assessing the specific levels of oxygenation levels at the entrance and exit of the microvascular system and allows assessing the specific levels of oxygen consumption in tissues. In particular, this approach allows revealing pathogenic processes resulting from hyper- and hypo-oxygenation in tissues. For instance, rapidly growing malignant tumors are characterized by intensive metabolism, rapid formation of capillaries, and active transcapillary oxygen exchange that results in higher level of oxygen diffusion into tissue, while the level of oxygen is lowered in the microvascular veins.

In the past two decades, the applications of optical monitoring for non-invasive assessment of glucose have been pursued with limited success. We have investigated potential application of optical coherence tomography (OCT) for non- invasive and continuous monitoring of blood glucose concentration. An OCT system with the wavelength of 1300 nm was used in phantom and in vivo studies. Polystyrene spheres with the diameter of 0.76 micrometers were used as scatterers in aqueous solutions in the phantom studies. We have found 4.5% change of the OCT signal slope as a function of glucose concentration in the range from 0 to 100 mM in the phantoms. This is in good agreement with theoretical calculations performed using Mie's theory. Bolus glucose injection and glucose clamping experiments were performed in New Zealand rabbits and Yucatan micropigs. OCT images were obtained from skin (dorsal area of the pigs and rabbit ear). Our pilot studies show close correlation between actual blood glucose concentration and slope of the OCT signals. The slope decreased substantially (about 40% in tissues in vivo) with the increase of blood glucose concentration from 4 to 30 mM. In conclusion, we have demonstrated that glucose-induced changes in optical properties of skin can be monitored by OCT suggesting that a new OCT-based optical sensor could be developed for sensitive and accurate non-invasive monitoring of glucose concentration in vivo.