A preliminary in vivo study using photopolymerized poly(ethylene glycol) (PEG) microspheres containing tetramethylrhodamine isothiocyanate labeld concanavalin A (TRITC-Con A) fluroescein isothiocyanate labeld dextran (FITX-dextran) as an implantable glucose sensor was performed using hairless rats. The glucose sensor works by affinity reaction between the two fluorescent labeled molecules binding together to form a fluorescent energy transfer system in which the FITC peak is quenched by the TRITC peak. The addition of glucose to the sensors local environment displces the dextran disrupting the FRET pair and the quenching. The change in fluroescent peak ratio (TRITC/FITC) therefore can be related to glucose. The microspheres in this study were implanted below the dermal skin layer of the lower abdomen by injection. A bolus injection of glucose was given through the tail vein to simulate glucose consumption. Spectra were obtained by shining and collecting light through the skin using an optical fiber delivery system via a 488nm argon laser and a spectrophometer. The preliminary results showed quantifiable changes in the ratio between the two peaks in response to the changae in glucose levels in the interstitial fluid of the rat.

Four-channel Kromoscopic analysis is demonstrated for in vitro measurement of glucose in cow blood in the spectral range of the first overtone of CH stretching vibrations and also the short wavelength near infrared region. The sample set included 48 blood samples with glucose concentration randomly distributed in the range 4.55 -37.60 mM. Solid glucose was added to each blood sample to create a wide range of glucose concentrations. During the measurement procedure blood was circulated through a 1 mm path length cell at a flow rate of 3 m:/min. Regular pulsations on the order of 1% were observed and corresponded to periodic aggregation and disaggregation of red blood cells under these flow conditions. Glucose can be quantified with a standard error of prediction of 1 mM over all blood samples. The impact of scattering and absorption proceses are also discussed.

The study on the magnitude of spectral change in blood glucose measurements with diffuse reflectance NIR spectroscopy is presented. Spectral change is estimated by a Monte Carlo simulation and measurements of absorbance spectra of aqueous glucose solution. Required sensitivity of spectrophotometers for monitoring the changein the blood glucose concentration as small as 10mg/dL has been obtained using the estimated change in the absorbance spectrum and mean pathlength of light in tissue.

Management of diabetic disease requires frequent monitoring of blood glucose concentration. Development of a noninvasive technique capable of reliable and sensitive monitoring of blood glucose concentration would considerably improve quality of life of diabetic patients and reduce mortality associated with this disease. Recently, we proposed to use Optical Coherence Tomography (OCT) technique for noninvasive glucose monitoring. In this paper, we tested in animals several aspects of specificity of noninvasive blood glucose monitoring with the OCT technique. Influence of temperature and tissue heterogeneity on the OCT signal profile is experimentally studied in this paper. We also theoretically investigated the changes in tissue scattering induced by variation of concentration of glucose and other osmolytes. Obtained results suggest that although several physical and chemical agents could potentially interfere with blood glucose concentration measurements using the OCT technique, their effect is smaller compared to that of glucose under normal physiological conditions.

A non-invasive biomedical sensor for monitoring glucose levels is described. The sensor utilizes laser light to determine glucose levels in urine, but could also be used for drug screening and diagnosis of other medical conditions. The glucose measurement is based on modulation spectroscopy with harmonic analysis. Active signal processing and filtering are used to increase the signal-to-noise ratio and decreases the measurement time to allow for real time sample analysis. Preliminary data are given which show the concentration of glucose in a control sample. Future applications of this technology, for example, as a portable multipurpose bio-medical analysis tool, are explored.

A Monte Carlo method was developed to model light transport through multi-layered tissue with the application focused on the development of an implantable perfusion monitor. The model was developed and then verified experimentally with a micro perfusion phantom. The program modeled a three-layer (tissue, capillary bed, tissue) scenario to investigate the source-detector separation effects for an implantable sensor. The Monte Carlo code was used specifically to model the effects of absorption and scattering properties of the surrounding tissue, the hemoglobin concentration in the middle layer, the ratio of thickness of the capillary layer to the first layer, and the probe-source separation distance on the propagation of the light through the tissue. The model was verified experimentally, using a simple in vitro system with optical source and detector fibers separated at various distances. The model was also used to investigate fluctuations in luminance as a result of hemoglobin concentrations and the response of the system to various wavelengths. The model was helpful for an ongoing project to develop an implantable perfusion monitor for transplanted organs or skin flaps.

In laser Doppler flowmetry (LDF) deep perfusion measurements can be realized by using a large separation between the fibers used for illumination and detection. In order to achieve a sufficient signal-to-noise ratio, the power of the laser light can be increased, but only to the limit indicated by the safety regulations. In this paper, pulsed laser Doppler flowmetry (pLDF) is presented as a manner to increase the SNR without exceeding the safety limits. The method is based on the principle that light is needed only when the signal is being sampled. The setup is presented, and we will show results that indicate that equivalent results are obtained for a pulsed and continuous wave setup (cwLDF), however with a much smaller tissue exposure. Furthermore, the limits encountered in realizing a pulsed system will be discussed.

The many biological and medical problems associated with microlymphatic functioning and its disturbances at different diseases, include primary and secondary lymphedema, inflammation, lymphatic malformations, and so on. It is important both to establish an adequate animal model for study lymph microcirculation in vivo and to match it with corresponding diagnostic techniques. The rat mesentery has been successfully used in experiments focusing on the microcirculation, including small lymphatics. Among optical methods the transmittance microscopy is most widely employed to study microcirculation. We have undertaken following investigations: development and evaluation of capability of transmission microscopy for in vivo studies of microcirculation; obtaining of single cell images; estimation of lymph microcirculation parameters, including the relation of forward to backward flow in intact lymphatics; regulation of microlymphatic function by nitric oxide and study of microlymphatic disturbances at the experimental lymphedema. Although interesting data has been obtained, the transmission microscopy has the relatively low absorption sensitivity and prevents obtaining good absorption contrast. To obtain more comprehensive physiological data, the further development and improvement imaging of rat mesentery is discussed with focus on new combined optical imaging systems which integrate recent advances in video-transmission and photothermal (PT) microscopy, PT fluid velocimetry, and laser spectroscopy.

The purpose of the present study is to investigate systematically the mechanisms of alterations in the optical properties of whole blood immersed in the biocompatible agent dextrans, and to define the optimal concentration of dextrans required for blood optical clearing in order to enhance the capability of light penetration depth for optical imaging applications. In the experiments, dextrans with different molecular weights and various concentrations were employed and investigated by the use of the optical coherence tomography technique. Changes in light attenuation, refractive index and aggregation properties of blood immersed in dextrans were studied. It was concluded from the results that the mechanisms for blood optical clearing are the characteristics of the types of dextrans employed, their concentrations, and the application stages. Among the substances applied, DX500 at a concentration at 0.5gdl^-1 gives the best result in improving light penetration depth through the blood. The increase of light transmission at the beginning of the addition of dextrans is mainly attributed to refractive index matching between the scattering centers and the ground matter. Thereafter, the transmission change is probably due to dextran-induced aggregation-disaggregation effect. Overall, light scattering in the blood could be effectively reduced by the application of dextrans. It represents a promising approach to increasing the imaging depth for in vivo optical imaging of biological tissue, for example optical coherence tomography.

The aim of this study was to measure the velocity and velocity profile of 0.3% Intralipid mixture in a 1.5-mm thick glass capillary using Doppler Optical Coherence Tomography (DOCT). First, while still empty, the dimensions of the capillary were measured. The outer diameter was 1.50 mm ± 0.01 mm while the lumen diameter was 1.01 ± 0.01 mm. Then, having filled the capillary with 0.3% solution, the lumen diameter was measured again. The mean refractive index of the solution was calculated and turned out to be 1.36 ± 0.01 mm. During the next stage, flow measurements were performed at an angle of 88° between the illuminating beam and the velocity vector of the fluid. The velocity profile, based on a set of 10 measurements, was calculated from the DOCT signal using a discrete Fourier transform in 32 sections of the capillary. The maximum velocity, located in the middle part of the capillary, was 0.256 ± 0.035 m/s. The results show that the flow velocity profile of 0.3% Intralipid solution can be obtained from a glass capillary.

Optical transmission and light scattering measurements were performed on three species of bacteria with different sizes and shapes (Pseudomonas aeruginosa. Staphylococcus aureus and Bacillus subtilis). The average bacteria size was determined from transmission measurements by using the Gaussian Ray Approximation of Anomalous Diffraction Theory. A rescaled spectra combining multiple angular data was analyzed in the framework of the Rayleigh-Gans theory of light scattering in order to determine particle shape and size distribution. Particle size and shape as determined by both methods are in good agreement with size and shape measured by scanning electron microscopy. These results demonstrate that light scattering may be able to detect and identify microbial contamination in the environment.

The ability to measure changes in bone-mineral-density (BMD) in-vivo has potential applications in monitoring stress-induced bone remodelling in, for example, competition race horses. In this study we have begun to investigate the potential of optical techniques to monitor such changes via changes in bone optical scattering. Using integrating spheres, we have investigated the optical properties of bone samples taken from the leg of the horse. Since our samples have stable characteristics over the time, we are able to use a single integrating-sphere technique. Diffuse reflection and transmission coefficients have been measured over the wavelength range 520 to 960 nm. Measurements were made on samples immersed in formic acid solution for different lengths of time; this was to investigate the effect of reduction in BMD on the optical properties. The experimental results and a Monte-Carlo based inversion method were used to extract the absorption coefficient and unmodified scattering coefficient of the samples. After full demineralisation scattering coefficient fell by a factor 4. This shows that the calcium-content in bone influences its optical properties considerably. Our experiments confirm the possibility of using optical techniques to determine changes in the BMD of samples.

A two wavelengths CO₂ laser was used to determine the glucose concentration in whole blood samples. For this purpose, the first laser wavelength λ₁ was tuned to the maximum of the glucose absorption band at 1080 cm^-1 and the second laser wavelength λ₂ was tuned to the background close to the glucose absorption band at 950 cm^-1. The difference of the laser power λ₁ and λ₂ behind the optical cell containing the whole blood sample was determined. This signal correlates strongly with the glucose concentration in the whole blood samples. The results of the investigation were compared to the results obtained by FTIR measurements.

The effect of changing corneal birefringence, due to motion artifact, remains a major obstacle to the development of an accurate non-invasive polarimetric glucose sensor for patients with diabetes mellitus. Consequently, there is still a need to characterize fully, and to quantify the relative changes in corneal birefringence to facilitate the optimization of detection algorithms, enabling in vivo accuracy within 10mg/dl. In this paper, we present preliminary results, utilizing a Mueller matrix imaging technique, that demonstrates notable relative changes in the apparent retardance and in the apparent fast axis location of rabbit cornea.

Traditional Raman spectroscopy while extremely sensitive to structure and conformation, is an ineffective tool for the detection of bioanalytes at the sub milimolar level. Surface Enhanced Raman Spectroscopy (SERS) is a technique developed more recently that has been used with applaudable success to enhance the Raman cross-section of a molecule by factors of 10⁶ to 10¹⁴. This technique can be exploited in a nanoscale biosensor for the detection of pathogenic proteins and DNA in foods by using a biorecognition molecule to bring a target analyte in close proximity to the mental surface. This is expected to produce a SERS signal of the target analyte, thus making it possible to easily discriminate between the target analyte and possible confounders. In order for the sensor to be effective, the Raman spectra of the target analyte would have to be distinct from that of the biorecognition molecule, as both would be in close proximity to the metal surface and thus be subjected to the SERS effect. In our preliminary studies we have successfully used citrate reduced silver colloidal particles to obtain unique SERS spectra of α-helical and β-sheet bovine serum albumin (BSA) that served as models of an α helical antiobiody (biorecognition element) and a β-sheet target protein (pathogenic prion). In addition, the unique SERS spectra of double stranded and single stranded DNA were also obtained where the single stranded DNA served as the model for the biorecognition element and the double stranded DNA served as themodel for the DNA probe/target hybrid. This provides a confirmation of the feasibility of the method which opens opportunities for potentially wide spread applications in the detection of food pathogens, biowarefare agents, andother bio-analytes.

In this paper we present theoretical analysis to support the polarimetric approach for glucose detection in the human eye applying Brewster reflection off the ocular lens. The theoretical eye model of Navarro, which is based upon anatomical data, was used to perform ray-tracing, whereas the electromagnetic and polarization parameters of light propagation through the eye-media were calculated. The errors in glucose concentration determination due to refraction and deviation from the ideal optical path were calculated under different conditions. Effects of using incident linearly and circularly polarized light and variation of intersection condition of the incoming light beam with the anterior corneal surface were taken into consideration. Calculations were performed for a wide spectral range by applying dispersion curves for the eye-media. These simulations show the potential and the limits of the proposed optical approach.

Native fluorescence emission and excitation spectra were used to monitor changes in Bacillus subtilis (Bs) and Staphylococcus aureus (Sa) subjected to starvation conditions. Initially, the fluorescence spectra from the Bs and Sa was dominated by tryptophan emission. After the second day, a fluorescence band with an emission peak at 410 nm and an excitation peak at 345 nm appeared in the Bs. This emission is from dipicolinic acid, a major constituent of bacterial endospores. The dipicolinic acid intensity increased steadily during the next 2 to 4 days as the number of Bs forming spores increased. No dipicolinic acid signal was observed in the (non-spore forming) Sa. The addition of β-hydroxybutyric acid to either the Bs or Sa resulted in the emergence of a third band with very strong fluorescence emission maximum at 460 nm and with excitation maxima at 250, 270 and 400 nm. This 460 nm emission was quenched with the addition of Fe²⁺, indicating that the source of this emission is a siderophore produced by the bacteria.

A series of experiments were performed to assess the feasibility of measuring total hemoglobin (Hb_T) non-invasively using light in the visible to near infrared (NIR) range. The experiments included spectroscopic measurements of both in vitro isolated tissue components and in vivo human tissue (73 subjects) in a clinical setting. Several different methods of estimating Hb_T, all by optical non-invasive means, were tested and compared for accuracy relative to standard invasive methods. By combining time-resolved optical absorbances at 3 wavelengths, 750, 965, and 1320 nm, in a fashion that is highly analogous to pulse oximetry, Hb_T was predicted with a standard error of 1.1 g/dL.

Ellipsometry is well known for its extreme sensitivity to the presence and properties of ultra-thin films. In the infrared, resonance response to chemical bonds allows chemical identification in monolayer-thick biological films. In this paper we show results of attachment repeatability for successive layers of monosialoganglioside, cholera toxin, and related antibodies using in situ visible spectroscopic ellipsometry. Several factors contributing to difficulty in obtaining reproducible results are discussed. Soecifically, these include freshness of reagents; surface type, cleaning, and preparation; temperature; birefringence of liquid cell windows; and cell design. Sensitivity and signal noise considerations for infrared spectra of molecular monolayers are discussed.

The non-invasive optical technique of dynamic light scattering (DLS) is routinely used to characterize dilute and transparent sub-micron particle dispersions in laboratory environments. A variety of industrial and biological applications would however greatly benefit from on-line monitoring of dispersions under flowing conditions. In this paper we present a model experiment to study flowing dispersions of polystyrene latex particles of varying sizes under varying flow conditions using a newly developed fiber optic DLS probe. A modified correlation function proposed in an earlier study by Chowdhury et al. is applied to the analysis of extracting size and velocity of laminar flowing particulate dispersions. The complimentary technique of laser Doppler velocimetry is also used to measure the speed of moving particles to confirm the DLS findings.

Blood hemoglobin (Hb) level is an important health parameter for a large segment of the population. Low Hb can indicate anemia due to chemotherapy, HIV, alcoholism, internal bleeding or other blood loss. There is a great need for noninvasive Hb measurement. A total blood Hb measurement method is shown which does not disturb the subject's skin. Results were obtained using MinforMed's noninvasive blood analyzer prototype (patent pending). A light is shined onto a body part, through the skin, engaging the blood. The emerging light is analyzed for Hb's signature strength in the visible and infrared ranges. Orthogonal decomposition methods are used to extract the Hb data from the complete spectrum. Results were compared to a laboratory-grade instrument that uses a drop of blood. A Hb range from 11 g/dL through 19 g/dL shows excellent correlation, r²=0.97. Other characteristics of the complete spectrum give indication of additional blood analytes, most notably bilirubin and water. Initial results are also shown indicating how light scattering varies with Hb concentration. Approximate residual skin and tissue spectrum is found by removing the spectral signature of the four Hb components (oxy-Hb, deoxy-Hb, carboxy-Hb and met-Hb) from the complete spectrum. This procedure yields the least squares concentrations of the individual Hb components. An SBIR grant from NIH is currently in progress on related work.

Background: Currently clinical Doppler ultrasound cannot detect microvascular blood flow and it is difficult to provide depth discrimination using laser Doppler flowmetry. Doppler optical coherence tomography (DOCT) is a novel technique for noninvasive subsurface imaging of microcirculation and tissue structure. Aims: To design handheld and catheter-based DOCT probes for clinical cutaneous and endoscopic imaging. To develop signal processing techniques for real-time detection and quantification of microvascular blood flow. Methods: A DOCT system, with interchangeable cutaneous and catheter probes, was developed. The axial spatial resolution was 10 μm, and the velocity resolution was 20 μm/s, using a 1300 nm broadband infrared light. The system achieved real-time imaging with frame rates up to 32 Hz at 512 x 256 pixels per frame. We used the system to detect microcirculation in human skin and rat esophagus, and to monitor microvascular responses to photodynamic therapy (PDT) in a rat tumor model. Results: We present experimental results from in vivo DOCT imaging of microcirculation in human skin arterio-venous malformations (AVM), normal rat esophagus, and a rat gliosarcoma PDT model. In the PDT model, we followed microvascular responses to PDT and observed differences in the microcirculation during and after therapy, which can have important implications for PDT dosimetry and treatment optimization. Conclusions: To our knowledge, this is the first demonstration of endoscopic catheter-based DOCT detection of microcirculation in vivo. In addition, AVM can be detected using handheld cutaneous DOCT probes under clinical settings. DOCT may serve as a real-time monitoring tool for PDT dosimetry, especially for vascular targeting photosensitizers.