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     

Fluorophore quantitation in tissue-simulating media with confocaldetection

Fluorescence measurements from tissue are increasingly being used as a medical diagnostic procedure to assess tissue malignancy or tissue function. Unfortunately, the reemitted fluorescent intensity measured from a tissue surface is not necessarily proportional to the fluorophore concentration because the light is altered by the tissue's intrinsic absorption and scattering properties. By measuring fluorescence from tissue volumes which are smaller than the average scattering length, the effects of the tissue's intrinsic absorption are diminished. In this study, experiments with tissue simulating phantoms are used, as well as Monte Carlo simulations of the experiment, to demonstrate the utility of point fluorescence detection for diagnostic measurements. Potential applications of this technique range from photosensitizer quantitation in vivo, pharmacokinetic measurements of fluorophore in different tissues, to any application where fluorophore quantification is required from a highly scattering medium     

Elastic Scattering Spectroscopy as a Diagnostic Tool for Apoptosis in Cell Cultures

Apoptosis, ldquoprogrammed cell death,rdquo is a cellular process exhibiting distinct biochemical and morphological changes. There is much interest regarding the role of apoptosis in cancer and the response to cancer treatment. Although apoptosis can occur spontaneously in malignant tumors and often significantly retards their growth, the initial response to successful cancer treatment is often massive apoptosis. In typical in vitro studies, current apoptosis detection methods require cell culture disruption via fixation, trypsinization, and/or staining. Our aim is to develop a nondisruptive optical method of detecting and tracking apoptosis in living cells and tissues, initially focusing on cell cultures. Such a method would allow for real-time evaluation of apoptotic progression of the same cell culture over time without perturbation or alteration. We report initial studies on the use of in vitro elastic scattering spectroscopy (ESS) to monitor changes in light-scattering properties of cells due to apoptotic morphology changes. For a sequence of times post treatment, we have measured the angle-dependent scattering at a single wavelength and also the wavelength-dependent scattering at discrete angles, of treated and control cell cultures. A novel polar nephelometer, developed in our laboratory, was used to obtain the angle-dependent scattering for the range of 90-145. Wavelength-dependent ESS measurements were made with a spectrometer, for several discrete near-forward angles. The results indicate that light scattering measurements can reliably discriminate between treated and control cells, correlating well with benchmark assays for apoptosis.     

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.     

Characterization of Fluorescence Lifetime of Photofrin and Delta-Aminolevulinic Acid Induced Protoporphyrin IX in Living Cells Using Single- and Two-Photon Excitation

Photodynamic therapy (PDT) is an effective treatment option for various types of invasive tumors. The efficacy of PDT treatment depends strongly on selective cell uptake and selective excitation of the tumor. The characterization of fluorescence lifetimes of photosensitizers localized inside living cells may provide the basis for further investigation of in vivo PDT dosage measurements using time-domain spectroscopy and imaging. In this communication, we investigated the fluorescence lifetime of localized Photofrin and delta-aminolevulinic acid (ALA) induced protoporphyrin IX (PpIX) in living MAT-LyLu (MLL) rat prostate adenocarcinoma cells. The MLL cells were incubated with the photosensitizers, and then treated with light under well-oxygenated conditions using a two-photon fluorescence lifetime imaging microscope (FLIM). Fluorescence lifetime images of these cells were recorded with average lifetimes of 5.5 plusmn 1.2 ns for Photofrin and 6.3 plusmn 1.2 ns for ALA-induced PpIX. When localized in cells, the lifetimes of both photosensitizers were found to be significantly shorter than those measured in organic solutions. The result for PpIX is consistent with literature values, while the lifetime of Photofrin is shorter than what has been reported. These results suggest that time-domain methods measuring photosensitizer lifetime changes may be good candidates for in vivo PDT dosage monitoring.     

Polarized light scattering spectroscopy for quantitativemeasurement of epithelial cellular structures in situ

We report an in situ method of probing the structure of living epithelial cells, based on light scattering spectroscopy with polarized light. The method makes it possible to distinguish between single backscattering from uppermost epithelial cells and multiply scattered light. The spectrum of the single backscattering component can be further analyzed to provide histological information about the epithelial cells such as the size distribution of the cell nuclei and their refractive index. These are valuable quantities' to detect and diagnose precancerous changes in human tissues     

Biological applications of near-field optical microscopy

Presents several biological applications of near field optical microscopy, in combination with force microscopy. Aperture near field scanning optical microscopy (NSOM) with fluorescence detection gives (bio)chemical specificity and orientational information, in addition to the simultaneously acquired force image. This technique has large potential for DNA sequencing, molecular organization in monolayers, and study of the role of the cytoskeleton in cellular mobility in cell growth, cell migration, formation of protrusions, etc. Fluorescence NSOM gives high resolution on flat, not too deep surfaces. Fluorescence NSOM induces virtually no bleaching, as opposed to confocal fluorescence microscopy. Bright field NSOM in transmission generally yields a complicated contrast, caused by a mixture of dielectric and topographic contributions. Shear force feedback is essential in aperture NSOM operation with fibers, and operates on soft surfaces of cells and chromosomes. Ultimately, aperture NSOM is limited by low efficiency with a source brightness of typically 100 pW to 10 nW. Thus, in spectroscopic applications (fluorescence, Raman, etc.) photon noise will be a fundamental limit in the speed of imaging. Photon tunneling in combination with force microscopy allows routine scanning with a high optical lateral resolution. However, interference effects can be dominant on surfaces which display extensive scattering. As such, the application potential of PSTM to biological surfaces is rather limited. Clearly, the virtues of optics, non-invasiveness, high spectral resolution, and high time resolution all apply to the near field optical domain with its high spatial resolution, which adds extensively to the potential of scanning probe microscopy     

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.     

The effect of light losses in double integrating spheres on opticalproperties estimation

The double integrating sphere setup (DIS) measures diffuse reflectance, diffuse transmittance and collimated transmittance, from which the optical properties of tissue (the absorption coefficient μ _a, the scattering coefficient μ_s and the anisotropy of scattering g) are estimated. The effect of light losses in the DIS and optical thickness on optical properties estimation by the inverse adding doubling algorithm (IAD) and uniqueness of measurement have been investigated using a Monte Carlo method. Results were obtained for optical properties in turbid tissues (0.80⩽albedo⩽0.99, 0.80⩽anisotropy⩽ 0.99, 1.5⩽optical thickness⩽7.5) sandwiched between glass slides. At optical thickness=6.0 the loss of light through the glass slides is ranging from 13%±0.5% (at albedo=0.80) to 15%±0.5% (at albedo=0.98) of the incident power. The loss of light at the exit port in the transmittance sphere is increasing up to 50% of the incident power at highly forward scattering. These losses result in a dependency on optical thickness of the optical properties estimation by the IAD algorithm. Furthermore, because of these losses, the DIS setup measurement is found to be fundamentally nonunique, when simultaneously measuring the diffuse reflectance, diffuse transmittance and collimated transmittance     

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     

Investigation of multidrug resistance in cultured human renal cell carcinoma cells by 31P-NMR spectroscopy and treatment survival assays

KTCTL-26 and KTCTL-2 are renal cell carcinoma (RCC) lines with high and lowexpression of P-170 glycoprotein, respectively. Inherent differences between the two cell lines in terms of phosphate metabolites and growth characteristics in culture were examined for possible association with multidrug resistance (MDR). Differences in response to drug treatment were investigated for 40 h incubations with various doses of vinblastine (VBL) alone or as cotreatments with various concentrations of the calcium antagonist diltiazem (DIL) and/or interferon–α (IFN-α). Treatment effects were quantitated using the MTT survival assay and ³¹P magnetic resonance spectroscopy (MRS) to determine phosphate metabolite profiles in intact cells. KTCTL-2 and KTCTL-26 cells exhibited significant inherent differences in phosphocholine, glycerophosphocholine, glycerophosphoethanolamine, and phosphocreatine levels. KTCTL-26 cells were more sensitive than KTCTL-2 to 0.011μM VBL alone (87% vs. 102% survival) or to 0.011μM BL + 10μM DIL (55% vs. 80% survival). The latter treatment resulted in a significant decrease in the ratio of phosphocholine to glycerophosphocholine in KTCTL-26 cells but no significant changes in phosphate metabolites in KTCTL-2 cells. Metabolomic ³¹P MRS detects different metabolite profiles for RCC cell lines with different MDR phenotypes and may be useful for noninvasive characterization of tumors in a clinical setting.This revised version was published online in August 2005 with a corrected sequence of authors.     

Selective cell killing by microparticle absorption of pulsed laserradiation

The mechanism of interaction between subcellular pigment microparticles and short pulse laser radiation was investigated with nanosecond time-resolved microscopy and fluorescence microscopy. Stroboscopic illumination was used to capture images of transient events induced in cells by laser pulses. Fluorescence microscopy enabled assessment of cell damage using fluorescent probes. Short-lived intracellular cavitation bubbles were directly visualized within nanoseconds after laser irradiation. Microbubble expansion and implosion took place on the timescale of 0.1-1 μs and were confined entirely within pigmented cells with remarkable selectivity. Cells containing particles underwent cavitation and rapidly lost viability, while adjacent cells without particles remained viable. Nonpigmented cells can be targeted by labeling them with absorbing particles. Laser-pumped microparticles provide an efficient means of selective cell targeting     

Study of normal and malignant white blood cells by time domaindielectric spectroscopy

The aim of this paper is to present a comprehensive theoretical and experimental study by means of time domain dielectric spectrometer (TDDS) of static and dynamic dielectric properties of normal and malignant blood cells. The successful use of the TDDS method as a tool of human cell study required a special protocol and algorithms for all stages of cell preparation, measurements and data treatment. The routine developed in this study was used in the experimental analysis of nine lines of malignant, transformed and normal lymphocytes. It was shown that dielectric permittivity, capacitance and conductivity values of the cell membrane are higher for normal lymphocytes in comparison with malignant ones     

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     

Wavelet transform of breast tissue fluorescence spectra: a technique for diagnosis of tumors

Polarized fluorescence spectra of malignant, benign, and normal human breast tissues in the emission range of 500-700 nm, with an excitation wavelength of 488 nm, are analyzed through discrete wavelet transform. The multiresolution and localization properties of the wavelets are shown to be ideally suited for identifying characteristic features distinguishing these tissue types. Analysis of a number of data sets, belonging to both parallel and perpendicular polarized spectra, have led to several key distinctions between different tumors and corresponding normal breast tissues, revealing the usefulness of polarized fluorescence in the diagnosis of tumors. Wavelet transform also naturally leads to the dimensional reduction of the data set, in the form of low-pass coefficients, making it amenable for physical modeling.     

高压输电线路宽频散射响应高效求解方法

针对传统均匀采样模型参数估计（MBPE）技术求解高压输电线路宽频散射特性存在的精度低、可靠性差等问题,引入一种自适应能力强的新MBPE技术,与构建的自适应频率采样算法结合,提出了一种准确快速求解高压输电线路宽频散射特性的新方法。以IEEE中波广播研究频段和调幅广播收音台工作频段为例,分别建立高压输电线路散射场的直线与线-面混合模型,将矩量法、MBPE技术和自适应频率采样算法结合,确定自适应采样点散射场信息和Stoer-Bulirsch有理插值函数,实现高压输电线路散射场宽频响应预测。计算结果表明所提出的自适应MBPE技术具有较高精度与可靠性。     

Investigating up-conversion fluorescence of Phloxine B

The steady-state and time-resolved emission of Phloxine B was examined when excited with 90 fs pulses from a mode-locked Ti:sapphire laser. Above the excitation wavelength (775 nm), Phloxine B was found to display two-photon excitation fluorescence with estimated cross-section for excitation of 0.87×10^-49 cm⁴ s/photon, and fluorescence spectrum consistent with one-photon excitation. Over the excitation wavelength range from 590 to 650 nm, Phloxine B was found to display one-photon excitation up-conversion fluorescence. The up-conversion fluorescence of Phloxine B was confirmed by frequency-domain measurements. The intensity decays revealed different double-exponential intensity decays for one-photon excitation at 390 nm and 633 nm. The longer fluorescence lifetimes were observed with 633 nm excitation. Hence, anti-Stokes excitation of Phloxine B causes delay emission at shorter wavelengths. Time-resolved anisotropy decay measurements revealed similar correlation times, but different amplitudes, as has been observed previously for two- versus one-photon excitation     

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.     

基于径向偏振光束的微粒捕获与操控

因为空间变化的偏振分布及独特的聚焦特性,径向偏振光束在粒子捕获及操控方面有独特的应用价值。从理论和实验方面研究了基于径向偏振光束的微粒捕获与操控。首先,介绍了捕获力的计算方法,重点基于光线理论模型计算了径向偏振光束的轴向及横向捕获效率,并与切向偏振光和圆偏振光的捕获效率进行了比较;然后,基于倒置显微镜和空间光调制器搭建了光学捕获与操控系统,采用两种不同的成像物镜实现了对直径为10μm左右的酵母菌细胞及直径为1μm的苯乙烯小球的捕获和操控,根据预定的轨迹实现了粒子的稳定移动,体现了该类型光镊较为宽阔的应用前景;最后,简要分析了影响粒子捕获及操控的若干因素,为系统改进提供了指导意见。