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.     

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.     

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.     

Bacteria size determination by elastic light scattering

Light extinction and angular scattering measurements were performed on three species of bacteria with different sizes and shapes ( Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis). The Gaussian ray approximation of anomalous diffraction theory was used to determine the average bacteria size from transmission measurements. A rescaled spectra combining multiple angular data was analyzed in the framework of the Rayleigh-Gans theory of light scattering. Particle shape and size distribution is then obtained from the rescale spectra. Particle characteristics (size and/or shape) retrieved from both methods are in good agreement with size and shape measured under scanning electron microscopy. These results demonstrate that light scattering may be able to detect and identify microbial contamination in the environment.     

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     

Spectroscopic imaging and the characterization of the autofluorescence properties of human bronchus tissues using UV laser diodes

Ultraviolet laser diodes (UV-LD) were used for the excitation source of autofluorescence (AF) measurements and spectroscopic imaging of the AF originating from the human bronchus was obtained. The AF spectra from normal bronchus tissues were measured and a clear AF spectrum was obtained by using a short wavelength (400 nm) laser diode; the overlap of the AF signal and excitation source could be substantially eliminated. In order to study the origin of AF intensity deterioration from bronchus tissue due to the formation of tumor tissues, the fluorescence spectrum was measured for various AF substances under various conditions. The blue AF signal of elastin and NADH solutions, which could not be easily studied by conventional excitation light sources, as well as the green AF became weak by adding lactic acid. The AF spectrum was measured for 512/spl times/512 pixels and the intensity mapping as a function of emission wavelength was obtained. Two-dimensional information of the AF signal intensity distribution for a certain wavelength component was measured. The feature originating from a region as small as about 100 /spl mu/m could be recognized. Numerical calculations of the data were performed and precise features of the AF were revealed.     

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     

Quantitative biodistribution and pharmacokinetics of multimodal gadolinium-based nanoparticles for lungs using ultrashort TE MRI

Objective

To study the biodistribution and lung pharmacokinetics of tracheally administered gadolinium-based contrast agents [gadoteric acid and multimodal ultra-small rigid platforms (USRPs)], to validate their pharmacokinetics against optical imaging of fluorescent USRPs, and to test their short-term toxicity.

Materials and methods

Ultrashort echo-time (UTE) lung proton magnetic resonance imaging (MRI) was performed at 4.7-Tesla (T) after the intratracheal instillation of different concentrations of contrast agent solutions in mice. Pharmacokinetic models were implemented on the absolute concentration calculated from the MRI signal enhancement measurements. Fluorescent USRPs were used to obtain optical images with the same protocol. Bronchoalveolar lavage inflammatory cell count and serum creatinine measurement were performed on four groups of instilled mice (sham, saline, USRPs, lipopolysaccharide).

Results

MR and optical imaging showed similar kinetics of the USRPs, passing from the airways to the lung tissue and to the kidneys, with negligible hepatic clearance. No significant increase of lung and renal inflammation markers were observed in USRP-instilled animals.

Conclusion

A T ₁-weighted radial UTE sequence was found to be valuable in quantitatively monitoring the biodistribution and pharmacokinetics of nanoparticles in the lungs of mice. The observed favorable pharmacokinetics, which was validated by fluorescence imaging, ensures the negligible toxicity of the nanoprobes, making the USRPs and the developed protocol good candidates for applications on selected lung diseases.     

Digital correction method for an H-field sensor in power system EMC measurements

Analysis was conducted on the nature and characteristics of an H-field sensor for power system electromagnetic compatibility (EMC) measurements. Based on the original calibrated response curve of the magnetic-field (H) sensor and steepest descent theory, an analog model has been created to represent the sensor's transfer function. This function was converted into a set of digital algorithms by the bilinear transformation method, thus allowing subsequent signal processing on available software. The digital correction model for the sensor gives an estimated error of within 8% for all the relevant frequency components. The results present some general analysis methods for power system EMC measurements.     

宽带PLC信号单分形和多重分形特性研究

宽带电力线通信(PLC)作为智能电网数据传输的有效途径,传统的线性模型和统计学参数难以描述电力线通信信号的非平稳、非线性特性缺点。为了更好地研究电力线通信信号特性,引入单分形和多重分形理论来分析宽带电力线通信信号的自相似特性。通过重标极差分析、变量时间图及周期图分析和小波改进理论等四种方法进行非线性特性分析,同时对不同频率和次数的分形分析方法进一步验证,结果表明宽带电力线通信信号存在自相似特性。此外,通过多重分形消除趋势波动分析法对宽带电力线通信信号进行单分形和多重分析特性测试,从实测的宽带电力线通信信号中估计了功率低指数的多重分形谱,同时提出了一种基于改进小波理论的多重分形消除趋势波动分析算法。     

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     

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     

Four methods to estimate the fractal dimension from self-affinesignals [medical application]

Four methods for estimating the fractal dimension, namely, relative dispersion, correlation, rescaled range, and Fourier (spectral) analysis, are described. Modifications of these methods for use on self-similar or self-affine signals are presented. It is found that correlation analysis and rescaled range analysis yield seriously biased results under many circumstances. Relative dispersion analysis is well suited for long signals. Spectral analysis gives the least biased results, and also has lowest variance in the estimates of the fractal dimension     

Glucose monitoring using implanted fluorescent microspheres

The authors are working toward a minimally invasive means for diabetics to better monitor glucose levels. A fiber-optic probe was fabricated for delivery and collection of light to an implanted sensor. The probe and associated optical system has high sensitivity and allows for flexible, remote measurements. The system was used to collect data with sensor solutions similar to what is envisioned for the implantable system, and the recorded spectra were processed in an attempt to predict glucose concentrations from fluorescence measurements. Despite the availability of sophisticated multivariate calibration methods, the best prediction results were obtained using polynomial regression with regression coefficient ratios. The measurements are far from perfect, but they do meet the requirements for clinical use and show promise for further improvement in the future. In vivo experiments were conducted using "simulated" sensors that had constant fluorescence characteristics. Results of implantation in excised skin and in live animals indicate that, while very strong fluorescence signals were measured for implanted particles, the current sensor chemistry must be improved for adequate resolution. In addition, difficulty in achieving repeatable injection depths and homogeneity of mixtures containing different colored spheres confounded attempts to make true quantitative measurements.     

Extinction coefficients of hemoglobin for near-infrared spectroscopy of tissue.

Extinction coefficients of hemoglobin have been studied for five decades by clinical chemists and biochemists, particularly for laboratory spectrophotometric measurements. In the last ten to 15 years, near infrared spectroscopy (NIRS) and imaging for tissue vascular oxygenation, breast tumor detection, and functional brain imaging have been intensively developed for in vivo measurements by groups of physicists, biomedical engineers, and mathematicians. In the approach of NIRS, NIR light in the wavelength range of 650-900 nm is utilized to illuminate tissue in vivo, and the transmitted or reflected light through tissue is recorded for the quantification of hemoglobin concentrations of the measured tissue vasculature. In order to achieve mathematical conversion from the detected light intensity at different wavelengths to hemoglobin concentration, extinction coefficients of hemoglobin, /spl epsiv/, must be used. While the engineers and physicists working in the NIR field have found the correct /spl epsiv/ values to use, there has been controversy on what /spl epsiv/ values should be used for in vivo NIRS in comparison with the conventional e/spl epsiv/ that most biochemists have used in the laboratories for in vitro measurements. The purpose of this article is to address this issue and help biomedical engineers and physicists gain a better understanding of e to be used for NIRS and NIR imaging.     

金属磁记忆检测中应力集中区信号的识别

金属磁记忆技术可以快速检测出铁磁构件的应力集中区,对其故障进行早期诊断。磁记忆信号非常微弱,容易受到外来噪声和干扰影响,使得难以准确确定应力集中区。针对含有噪声非平稳性的漏磁信号,在原有信号特征量梯度的基础上,给出了新的时域空间梯度和峰-峰值组合特征量,以及能量峰值处理方法,有效消除了随机噪声对磁记忆信号的影响,提高了应力集中区的识别率;从磁记忆信号的奇异性检测角度出发,通过多尺度小波系数的平方相关一致性来确定真正的应力集中区;实验验证了所提方法的有效性。     

Theory of design parameters for quasi-phase-matched waveguides andapplication to frequency doubling in polymer waveguides

A theory of dispersion in single-mode symmetric waveguides is presented for phase-matching second harmonic generation (SHG) of the fundamental modes, based on the approximate analytical waveguide theory of Botez. The theory is used to derive new equations for the maximum phase-matching distance allowed when there are random fluctuations in waveguide thicknesses, under critical and noncritical phase-matching conditions. The theory is also used to calculate the overlap integral as a function of the waveguide parameters V^ω and V^2ω. A new expression is derived for the efficiency of SHG in waveguides in terms of waveguide parameters that can be used to optimize SHG. Theoretical results are presented for typical LiNbO₃ and polymer waveguides. Quasi-phase-matched (QPM) waveguides are fabricated from nonlinear optical (NLO) polymers using the techniques of periodic poling and bleaching, and channel waveguides are printed by the bleaching of the NLO polymers. The NLO polymers are characterized for their refractive indexes, optical loss, NLO coefficients, and bleaching characteristics. Phase-matched SHG results are presented for the different fabrication methods over a distance of 0.5 cm, and an assessment is given of the relative strengths and weaknesses of the different fabrication approaches     

Indirect evidence for the potential ability of magnetic resonance imaging to evaluate the myocardial iron content in patients with transfusional iron overload

The purpose of this study was to evaluate the potential ability of magnetic resonance imaging (MRI) for evaluation of myocardial iron deposits. The applied MRI technique has earlier been validated for quantitative determination of the liver iron concentration. The method involves cardiac gating and may, therefore, also be used for simultaneous evaluation of myocardial iron. The tissue signal intensities were measured from spin echo images and the myocardium muscle signal intensity ratio was determined. The SI ratio was converted to tissue iron concentration values based on a modified calibration curve from the liver model. The crucial steps of the method were optimized; i.e. recognition and selection of the myocardial slice for analysis and positioning of the regions of interest (ROIs) within the myocardium and the skeletal muscle. This made the myocardial MRI measurements sufficiently reproducible. We applied this method in 41 multiply transfused patients. Our data demonstrate significant positive linear relationships between different iron store parameters and the MRI-derived myocardial iron concentration, which was significantly related to the serum ferritin concentration (ρ = 0.62.P < 0.0001) and to the MRI-determined liver iron concentration (ρ = 0.36,P = 0.02). The myocardial MRI iron concentrations demonstrated also a significant positive correlation with the number of blood units given (ρ = 0.45,P = 0.005) and the aminotransferase serum concentration (ρ = 0.54,P = 0.0008). Our data represents indirect evidence for the ability of MRI techniques based on myocardium/muscle signal intensity ratio measurements to evaluate myocardial iron overload.     

An Emerging Testing Method for Metallics Based on NV Center in the Diamond

Metal substance detection plays an extremely important role in daily life, industrial manufacturing and even industrial security. The traditional methods include optical detection, X-ray detection, microwave detection and ultrasonic detection. These methods, playing a vital role in the field of non-destructive testing, can not only judge the presence or absence of metal, but also accurately detect the type and size of metal defects. For mi-crowave detection, the detection efficiency of metal materials is limited by the response sensitivity of the de-tector to microwaves. In recent years, scientists have discovered a quantum sensing system based on the dia-mond nitrogen-vacancy (NV) color center. The system obtains optical detection magnetic resonance (ODMR) fluorescence spectra under the combined action of a 532nm laser and a certain frequency band of microwaves, and the signal contrast changes significantly with the microwave power. Based on the NV color center quantum sensing system, this paper studies its application in the field of metal detection, and takes steel detection as an example to detect the size of steel bars according to the changes in the spectral line, providing a new method for non-destructive testing such as metal substance detection.