Quantitative optical biopsy of liver tissue ex vivo

The intensity of the intrinsic autofluorescence of the reduced form of nicotineamide adenine dinucleotid (NADH) of biological tissue depends on the local, cellular concentration of this coenzyme. It plays a dominant role in the Krebs cycle and, therefore, serves as an indicator for the vitality of the observed cells. Due to the individually and locally varying boundary conditions and optical tissue properties, which are scattering coefficients, absorption coefficients and an anisotropy factor g, the fluorescence signal needs to be rescaled. Rescaling methods use for instance the Kubelka-Munk theory or the photon migration theory. Our rescaling method is partly based on measurements and partly theoretically derived. By combining four methods, i.e., laser-induced fluorescence (LIF) of the time-resolved signal, biochemical concentration measurements. Monte Carlo simulations with typical optical parameters and microscopic investigations, we demonstrate that simultaneous detection of the fluorescence and backscattering signal can easily and accurately provide rescaled, quantitative values for the NADH concentrations     

Inverse method 3-D reconstruction of localized in vivofluorescence-application to Sjogren syndrome

The development of specific fluorescently labeled cell surface markers have opened the possibility of specific and quantitative noninvasive diagnosis of tissue changes. We are developing a fluorescence scanning imaging system that can perform a “noninvasive optical biopsy” of the Sjogren syndrome (SS) which may replace the currently used histological biopsy. The diagnosis of SS is based on the quantification of the number of topical preadministered fluorescent antibodies which specifically bind to the lymphocytes infiltrating the minor salivary glands. We intend to scan the lower lip, and for each position of the scan, generate a two-dimensional (2-D) image of fluorescence using a charge-coupled device (CCD) camera. We have shown previously that our diffuse fluorescent photon migration theory predicts adequately the positions and strengths of one and two fluorescent targets embedded at different depths in tissue-like phantoms. An inverse reconstruction algorithm based on our theoretical findings has been written in C⁺⁺ and uses 2-D images to predict the strength and location of embedded fluorophores. However, due to large numbers of variables, which include the optical properties of the tissue at the excitation and emission wavelengths, and the positions and strengths of an unknown number of fluorophore targets, the validity of the final result depends on assumptions (such as the number of targets) and the input values for the optical parameters. Our results show that the number of fluorophore targets reconstructed for each scan is limited to two, and at least the scattering coefficient at the excitation wavelength is needed a priori to obtain good results. The latter can be obtained by measurements of spatially resolved diffuse reflectance at the excitation wavelength that provides the product of the absorption and scattering coefficients     

Determination of epithelial tissue scattering coefficient using confocal microscopy

Most models of light propagation through tissue assume the scattering properties of the various tissue layers are the same. The authors present evidence that the scattering coefficient of normal cervical epithelium is significantly lower than values previously reported for bulk epithelial tissue. They estimated the scattering coefficient of normal and precancerous cervical epithelium using measurements of the reflectance as a function of depth from confocal images. Reflectance measurements were taken from ex vivo cervical biopsies and fit to an exponential function based upon Beer's law attenuation. The mean scattering coefficients derived were 22 cm/sup -1/ for normal tissue and 69 cm/sup -1/ for precancerous tissue. These values are significantly lower than previously reported for bulk epithelial tissues and suggest that scattering of bulk tissue is dominated by the stroma. They also suggest that computational models to describe light propagation in epithelial tissue must incorporate different scattering coefficients for the epithelium and stroma. Further, the lower scattering of the epithelium suggests greater probing depths for fiber optic probes used by optical diagnostic devices which measure reflectance and fluorescence in epithelial tissue. The difference in scattering between normal and precancerous tissue is attributed to increased nuclear size, optical density, and chromatin texture. The scattering coefficients measured here are consistent with predictions of numerical solutions to Maxwell's equations for epithelial cell scattering.     

Effects of compression on soft tissue optical properties

Tissue optical properties are necessary parameters for prescribing light dosimetry in photomedicine. In many diagnostic or therapeutic applications where optical fiber probes are used, pressure is often applied to the tissue to reduce index mismatch and increase light transmittance. In this paper, we have measured in vitro optical properties as a function of pressure with a visible-IR spectrophotometer. A spectral range of 400-1800 mm with a spectral resolution of 5 nm was used for all measurements. Skin specimens of a Hispanic donor and two Caucasian donors were obtained from the tissue bank. Bovine aorta and sclera, and porcine sclera came from a local slaughter house. Each specimen, sandwiched between microscope slides, was compressed by a spring-loaded apparatus. Then diffuse reflectance and transmittance of each sample were measured at no load and at approximately 0.1, 1, and 2 kgf/cm². Under compression, tissue thicknesses were reduced up to 78%. Generally speaking, the reflectance decreased while the overall transmittance increased under compression. The absorption and reduced scattering coefficients were calculated using the inverse adding doubling method. Compared with the no-load controls, there was an increase in absorption and scattering coefficients among most of the compressed specimens     

Fluorescence Anisotropy of Cellular NADH as a Tool to Study Different Metabolic Properties of Human Melanocytes and Melanoma Cells

In this study, we wanted to see if fluorescence anisotropy could be used to detect changes in metabolism in cells with significant light scattering and absorption properties. Fluorescence anisotropy measurements of nicotinamide adenine dinucleotide (NADH) were performed with human melanocytes and melanoma cell lines. To demonstrate the feasibility of using fluorescence anisotropy for detecting metabolic changes, the electron transport chain was blocked using rotenone, inducing an accumulation of intracellular NADH. Total fluorescence increased in all cells as a result of rotenone treatment. Fluorescence anisotropy decreased in the rotenone-treated cells relative to the controls, suggesting an increased ratio of free to protein-bound NADH in the treated cells. In general, the fluorescence anisotropy of the melanocytes was significantly higher than that of the melanoma cell lines. Reflectance spectroscopy showed that the differences in fluorescence anisotropy between the cell types were not due to differences in scattering and absorption properties. Intrinsic cellular NADH fluorescence was experimentally extracted by ratioing polarized fluorescence to polarized reflectance. NADH binding, measured as the ratio of fluorescence intensity at 430 and 465 nm, showed more protein-bound NADH in the melanocytes than in the melanoma cells, consistent with the fluorescence anisotropy measurements.     

Sized-fiber reflectometry for measuring local optical properties

Sized-fiber spectroscopy describes a device and method for measuring absorption and reduced scattering properties of tissue using optical fibers with different diameters. The device used in this paper consists of two fibers with diameters of 200 and 600 μm. Each fiber emits and collects its own backscattered light. Backscattered light measurements for solutions with absorption coefficients of 0.1-2.0 cm ^-1 and reduced scattering coefficients of 5-50 cm^-1 demonstrate that the device is most sensitive for the highest scattering materials. Monte Carlo simulations suggest the device is insensitive to the fiber illumination characteristics and that the light returning to the fiber is nearly uniform over all directions. Finally, experiments and Monte Carlo simulations of the sized-fiber device indicate that 50% of the signal arises from roughly 1.2 and 1.9 reduced mean-free paths for the 200- and 600-μm fibers, respectively     

Single Gold Nanorod Detection Using Confocal Light Absorption and Scattering Spectroscopy

Gold nanorods have the potential to be employed as extremely bright molecular marker labels for fluorescence, absorption, or scattering imaging of living tissue. However, samples containing a large number of gold nanorods usually exhibit relatively wide spectral lines. This linewidth limits the use of the nanorods as effective molecular labels, since it would be rather difficult to image several types of nanorod markers simultaneously. In addition, the observed linewidth does not agree well with theoretical calculations, which predict significantly narrower absorption and scattering lines. The discrepancy could be explained by apparent broadening because of the contribution of nanorods with various sizes and aspect ratios. We measured native scattering spectra of single gold nanorods with the confocal light absorption and scattering spectroscopy system, and found that single gold nanorods have a narrow spectrum as predicted by the theory, which suggests that nanorod-based molecular markers with controlled narrow aspect ratios, and to a lesser degree size distributions, should provide spectral lines sufficiently narrow for effective biomedical imaging.     

An Alternative Approach to Analyze Fluorescence Lifetime Images as a Base for a Tumor Early Diagnosis System

Fluorescence lifetime imaging is a very promising imaging method for early detection of malignant tumors. It offers many advantages over conventional fluorescence methods, especially because the acquired signal does not rely on the fluorophore concentration in the tissue. As in all imaging method, the goal is to determine the exact location of a malignant tumor. However, since we are dealing with optical imaging, the inverse problem, i.e., extracting the tumor location coordinates is not an easy task to fulfill. In this paper, we describe an alternative method of interpreting the fluorescence lifetime image. The method extracts four features from each decay curve. We show that from these features one can extract the location of the tumor. The theoretical model is compared to the experimental results obtained from tissue-like phantoms.     

Modeling of transfer Characteristics for the broadband power line communication channel

This paper presents a novel approach to model the transfer function of electrical power lines for broadband power line communication. In this approach, the power line is approximated as a transmission line and the two intrinsic parameters, the characteristic impedance and the propagation constants, are derived based on the lumped-element circuit model. Using these intrinsic parameters, the transfer characteristics for a N-branch power distribution network are derived based on the scattering matrix method. Detail derivation of this line model is given in this paper. The model has been verified with practical measurements conducted on actual power networks. It is demonstrated that the model accurately determine the line characteristics under different network configuration and when different household appliances are connected.     

Toward nanometer-scale resolution in fluorescence microscopy using spectral self-interference

We introduce a new fluorescence microscopy technique that maps the axial position of a fluorophore with subnanometer precision. The interference of the emission of fluorophores in proximity to a reflecting surface results in fringes in the fluorescence spectrum that provide a unique signature of the axial position of the fluorophore. The nanometer sensitivity is demonstrated by measuring the height of a fluorescein monolayer covering a 12-nm step etched in silicon dioxide. In addition, the separation between fluorophores attached to the top or the bottom layer in a lipid bilayer film is determined. We further discuss extension of this microscopy technique to provide resolution of multiple layers spaced as closely as 10 nm for sparse systems.     

Accurate measurement of total attenuation coefficient of thin tissue with optical coherence tomography

Noninvasive accurate measurements of tissue optical properties are needed for many diagnostic and therapeutic applications. Optical coherence tomography (OCT) recently proposed for high-resolution imaging in tissue can potentially be applied for accurate, noninvasive, and high-resolution measurement of tissue total attenuation coefficient. However, confocal function (dependence of OCT sensitivity on the distance of probed site from the focal plane of the objective lens) and multiple scattering substantially limit the accuracy of the measurement with the OCT technique. We studied the influence of the confocal function and multiple scattering on the accuracy of the measurement and proposed methods that provide measurement of the total attenuation coefficient with a significantly reduced systematic error. Experiments were performed in tissue phantoms and porcine and human skin in vitro and in vivo. Our data indicate that the tissue total attenuation coefficient can noninvasively be measured in vivo with the accuracy of 5%-10% in the range from 0.5 to 17 mm/sup -1/ and about 20% in the range up to 40 mm/sup -1/. These results suggest that the proper correction of the OCT-based measurement for the confocal function and multiple scattering provides absolute values of tissue total attenuation coefficient with high accuracy and resolution that may not be achievable by other optical techniques in vivo.     

Optical Damage Limits to Pulse Energy From Fibers

The irradiances or fluences encountered in pulsed fiber lasers and amplifiers are sometimes high enough to destroy the fiber. The ultimate limit is set by the intrinsic damage threshold irradiance of doped silica. Other nonlinear processes such as self-focusing and stimulated Brillouin scattering are also important. We present here the results of our measurements of the damage threshold, and of our analysis of how self-focusing and Brillouin scattering influence the power limit.      

Frequency-domain optical detection of subsurface blood vessels:experimental and computational studies using a scattering phantom

A simple method to localize blood vessels beneath the surface of tissue could be very useful during laparoscopic and endoscopic procedures. However, the detection of blood vessels deep within tissue using light is limited by tissue scattering. In the study, frequency-domain photon migration methods were used to detect blood vessels within a scattering medium. The depth at which blood vessels could be detected was greater than 10 mm. The experimental measurements agree well with predictions obtained from the diffusion approximation to the radiative transport equation. These studies demonstrate the potential of frequency-domain optical methods to detect subsurface blood vessels     

Review and Recent Development of Angle-Resolved Low-Coherence Interferometry for Detection of Precancerous Cells in Human Esophageal Epithelium

The combination of low-coherence interferometry with angle-resolved light scattering measurements has been shown to be a powerful method for determining the structure of cell nuclei within intact tissue samples. The nuclear morphology data have been used as a biomarker of neoplastic change in a wide range of settings. Here, we review the development of angle-resolved low-coherence interferometry (a/LCI) for assessing the health status of human esophageal epithelial tissues based on depth-resolved measurements of the morphology of cell nuclei. The design and implementation of clinical instrumentation are reviewed, and results from ex vivo human tissue measurements are presented to validate the capabilities of the technique. In addition to the review of earlier papers, new results are presented, which demonstrate the first application of a portable a/LCI system with a flexible endoscopic probe to assessing depth-resolved nuclear morphology in a clinical setting. High sensitivity for the detection of precancerous tissues is demonstrated.     

An overview of activities at the Laser Biomedical Research Center

Research projects on laser heating and ablation and on spectroscopy of biological tissues are described. The discussion focuses on studies regarding microscopic laser light scattering of biological cells and structures, ablation of calcific tissue using pulsed HF laser radiation, and fluorescence and its use in diagnosing atherosclerosis. A specifically designed multifiber laser catheter constructed to collect tissue fluorescence spectra using fiber optics is described.     

Light propagation in tissue during fluorescence spectroscopy withsingle-fiber probes

Fluorescence spectroscopy systems designed for clinical use commonly employ fiberoptic probes to deliver excitation light to a tissue site and collect remitted fluorescence. Although a wide variety of probes have been implemented, there is little known about the influence of probe design on light propagation and the origin of detected signals. In this study, we examined the effect of optical fiber diameter, probe-tissue spacing and numerical aperture on light propagation during fluorescence spectroscopy with a single-fiber probe. A Monte Carlo model was used to simulate light transport in tissue. Two distinct sets of excitation-emission wavelength pairs were studied (337/450 nm and 400/630 nm). Simulation results indicated that increasing fiber diameter or fiber-tissue spacing increased the mean excitation-emission photon pair pathlength and produced a transition from high selectivity for superficial fluorophores to a more homogeneous probing with depth. Increasing numerical aperture caused an increase in signal intensity, but axial emission profiles and pathlengths were not significantly affected for numerical aperture values less than 0.8. Tissue optics mechanisms and implications for probe design are discussed. This study indicates that single-fiber probe parameters can strongly affect fluorescence detection and should be considered in the design of optical diagnostic devices     

Intraoperative application of optical spectroscopy in the presenceof blood

A simple but effective method of spectral processing was developed to minimize or remove the effects of the presence of superficial blood on tissue optical spectra and, hence, enhance the performance of optical-spectroscopic-based in vivo tissue diagnosis and surgical guidance. This spectral-processing algorithm was developed using the principles of absorption-induced light attenuation wherein the ratio of fluorescence intensity (F) and the hth power of diffuse reflectance intensity (Rd) at a given emission wavelength λ_m is immune to spectral distortions induced by the presence of blood on the tissue surface. Here, the exponent h is determined by the absorption coefficients of whole blood at the excitation and emission wavelengths. The theoretical basis of this spectral processing was verified using simulations and was experimentally validated. Furthermore, the optical spectra of brain tissues collected in vivo was processed using this algorithm to evaluate its impact on brain tissue differentiation using combined fluorescence and diffuse reflectance spectroscopy. Based on the simulation, as well as experimental results, it was observed that using F/Rd^h h can effectively reduce or remove spectral distortions induced by superficial blood contamination on tissue optical spectra. Thus, optical spectroscopy can also be used intraoperatively for applications such as surgical guidance of tumor resection     

Near-infrared (NIR) tomography breast image reconstruction with a priori structural information from MRI: algorithm development for reconstructing heterogeneities

A combined magnetic resonance and near-infrared (MRI-NIR) imaging modality can potentially yield high resolution maps of optical properties from noninvasive simultaneous measurement. The main disadvantage of near-infrared (NIR) tomography lies in the low spatial resolution resulting from the highly scattering nature of tissue for these wavelengths. MRI has achieved high resolution, but suffers from low specificity. In this study, NIR image reconstruction algorithms that incorporate a priori structural information provided by MRI are investigated in an attempt to optimize recovery of a simulated optical property distribution. The effect of high levels of tissue heterogeneity are evaluated to determine the limitations of incorporating prior information into a realistic set of patient breast images. We assume absorption coefficient (/spl mu//sub a/) variations near /spl plusmn/40%, and transport scattering coefficient (/spl mu//sub s//sup //) variations near /spl plusmn/20%, in a coronal breast MRI geometry. Changes in tissue pathology due to tumor growth can be observed with NIR tompgraphy, and so the goal here is to determine how best to quantify these tumor-based contrast regions within the presence of high tissue heterogeneity. By applying knowledge of tissue's layered structure in reconstruction through various constraints in the iterative algorithm, quantitative recovery of the tumor optical properties improves from 69% to 74%, and localization improves as well. However, only when the true heterogeneity of the tissue distribution was included was accurate quantification of the tumor region possible. Using a good initial guess of /spl mu//sub a/ and /spl mu//sub s//sup //, derived from the regional structure of the model, quantification of the region reaches 99% of the true value, and spatial resolution retains a similar value to the original MRI image.     

Electromagnetic Emission from Electric Propulsions under Ground Conditions

Analysis and methodological generalization of available methods used for determining characteristics of intrinsic emission from electric propulsions (EP) in a radio-frequency range that can be the interference for the “Earth-spacecraft (SC)” channel of the space communication system are the subjects of this paper. Intrinsic emission from the electric propulsion in a radio-frequency range is examined in detail by the example of a measuring complex developed in RIAME MAI and the measurement results are presented. The electric field intensity distribution in a radio-frequency range for the vertical and horizontal polarizations of the received emission is considered as the main characteristics. Measurements performed for the EP intrinsic emission by using the developed complex and measurements performed in metal vacuum chambers are compared and comparative results are presented in the paper.     

The mechanisms leading to the useful electrical properties of polymer nanodielectrics

Polymer nanocomposites with metal oxide nanoparticle fillers exhibit enhanced electrical breakdown strength and voltage endurance compared to their unfilled or micron filled counterparts. This paper presents the following hypothesis for the mechanisms leading to improved properties. The inclusion of nanoparticles provides myriad scattering obstacles and trap sites in the charge carriers' paths, effectively reducing carrier mobility and thus carrier energy. The result is homocharge buildup at the electrodes, which increases the voltage required for further charge injection due to blocking by the homocharge. The hypothesis is supported by electroluminescence, pulsed electro acoustic analysis, thermally stimulated current measurements, a comparison of AC, DC, and impulse breakdown, as well as absorption current measurements, in silica/crosslinked polyethylene matrix composites with supporting evidence from titania/epoxy composites.