Fluorescent Profiling of Venom-Selected Cobra Species

The aim of this paper was to characterize and compare spitting cobras of Africa. The autofluorescence of Naja ashei and Naja nigricollis venoms was investigated by atomic force microscopy and fluorescence analysis. The synchronous fluorescence analysis showed higher endogenous fluorescence of Naja ashei venom in comparison with Naja nigricollis venom. The atomic force microscopy revealed different surfaces of compared venoms as a result of different amounts of proteins. Our results confirmed different structures of Naja nigricollis and Naja ashei venom. The practical advantages of selected techniques are high sensitivity, minimal quantity of venom, and capability to measure the spitting cobra by autofluorescence.     

基于激光共焦扫描术的亚心形扁藻显微图像与光谱

采用激光共聚焦扫描显微技术,针对亚心形扁藻开展了研究.从获得的488 nm Ar+激光单光子激发的亚心形扁藻自体荧光光谱与图像,可知细胞内有一杯状叶绿体物质,其荧光峰值为682 nm,对应叶绿体发出的红色荧光.在单通道模式下,获得800 nm fs激光双光子激发的扁藻自体荧光光谱与图像,可知每个杯状叶绿体的内部有一个自体荧光更强的圆形物质.在双通道模式下,可分别获得小圆形物质的自体荧光图像,杯状叶绿体自体荧光图像,以及两个通道图像的叠加.进一步获得了双光子藻细胞荧光图的6个主要的荧光峰.采用单光子激光激发可获得亚心形扁藻叶绿体自体荧光图像及其荧光光谱,而双光子激光激发荧光光谱的多通道以及Lambda模式下采集光谱信号与图像,不仅可观察到亚心形扁藻的内部形态结构,还可能从双光子激发荧光图中研究分析亚心形扁藻生化物质的存在,灵敏度较单光子激发高.激光扫描共聚焦显微技术,特别是双光子荧光与图像技术可为海藻的检测与研究提供一种快速、实时、有效、简便的方法.     

Evaluating cutaneous photoaging by use of multiphoton fluorescence and second-harmonic generation microscopy

The photoaging process of facial skin is investigated by use of multiphoton fluorescence and second-harmonic generation (SHG) microscopy. We obtain the autofluorescence (AF) and SHG images of the superficial dermis from the facial skin of three patients aged 20, 40, and 70 years. The results show that areas of AF increase with age, whereas areas of SHG decrease with age. The results are consistent with the histological findings in which collagen is progressively replaced by elastic fibers. The AF and SHG changes in photoaging are quantified by a SHG to autofluorescence aging index of dermis (SAAID). Our results suggest that SAAID can be a good indicator of the severity of photoaging.     

Label-Free Spatial Analysis of Free and Enzyme-Bound NAD(P)H in the Presence of High Concentrations of Melanin

The analysis of autofluorescence, often regarded as undesired noise during the imaging of biological samples, allows label free, unbiased detection of NAD(P)H and melanin in native samples. Because both the emission and absorption spectra of these fluorophores overlap and they can hence not be differentiated using emission filters or with different excitation wavelengths, fluorescence lifetime imaging microscopy (FLIM) is used to differentiate between them. In the present paper the application of two-photon excitation microscopy is presented to investigate the autofluorescence of fungal spores. The model organism which was examined is Aspergillus ochraceus. Furthermore a strategy is developed which allows to quantitatively analyze the fluorescence lifetimes of melanin, free NAD(P)H and protein-bound NAD(P)H using forward convolution of a multiexponential decay function with the instrument response function (IRF) and subsequent fitting to the experimental fluorescence data. As a consequence proteins, which are able to bind NAD(P)H, are located with sub-cellular resolution. Furthermore a spatial differentiation of the fluorophores NAD(P)H and melanin inside the spores, is revealed.     

Delayed observation of laser-induced fluorescence for imaging of tumors

We report on a technique to improve fluorescence images of superficially growing tumors marked with photosensitizers. Exploiting the longer fluorescence decay times of porphyrin-based photosensitizers compared to average decay times of tissue autofluorescence, delayed detection of laser-induced fluorescence allows to suppress the autofluorescence background. The feasibility of delayed fluorescence imaging of tumors has been demonstrated in-vitro. It follows from an analysis of delayed fluorescence spectra that autofluorescence background falling into the photosensitizer fluorescence band can be reduced by up to one order of magnitude.     

Sensing cell metabolism by time-resolved autofluorescence

We built a time-resolved confocal fluorescence spectroscopy system equipped with the multichannel time-correlated single-photon-counting technique. The instrument provides a unique approach to study the fluorescence sensing of cell metabolism via analysis of the wavelength- and time-resolved intracellular autofluorescence. The experiments on monolayered cell cultures show that with UV excitation at 365 nm the time-resolved autofluorescence decays, dominated by free-bound reduced nicotinamide adenine dinucleotide signals, are sensitive indicators for cell metabolism. However, the sensitivity decreases with the increase of excitation wavelength possibly due to the interference from free-bound flavin adenine dinucleotide fluorescence. The results demonstrate that time-resolved autofluorescence can be potentially used as an important contrast mechanism to detect epithelial precancer.     

Numerical and experimental study of excited light and auto-fluorescence diffusion in teeth

Excited light and corresponding intrinsic fluorescence diffusion inside teeth tissue are an essential problem for light-based carious lesion detection. Based on finite element numerical analysis of diffusion equation, the photon density distribution of both excited light and autofluorescence of 2D premolar teeth model is obtained. The dependence of excited light and autofluorescence density distribution inside the teeth model on the scattering coefficient of enamel (5–25 mm⁻¹) and dentine (100–140 mm⁻¹) is numerically simulated and analyzed. The fitted results reveal that fluorescence intensity decreases exponentially. Optical penetration depth and fluorescence relative depth declined with the increment of scattering coefficient of enamel. And the dentine had the opposite effect. Finally, the experiment of measurement of fluorescence intensity on the teeth surface is conducted and the result is compared with the numerical computation.     

A Study of the Intrinsic Autofluorescence of Poly (ethylene glycol)-co-(<Subscript><Emphasis Type="Italic">L</Emphasis></Subscript>-Lactic acid) Diacrylate

Poly (ethylene glycol)-co-( _L -Lactic acid) diacrylate (PEG-PLLA-DA) copolymers have been extensively investigated for a number of applications in medicine. PEG-PLLA-DA is biodegradable and the human body can process its degradation products. In this study, we describe the autofluorescence of PEG-PLLA-DA copolymers and compared it to the fluorescence of poly(ethylene glycol) diacrylate (PEG-DA) and the precursor molecules used for their synthesis. In addition, we examined the influence of pH on the fluorescence spectra. We found that PEG-PLLA-DA exhibits higher fluorescence than PEG-DA and all reagents involved in the synthesis of PEG-PLLA-DA. The fluorescence of PEG-PLLA-DA was affected by pH with fluorescence decreasing at high pH values. At high pH, PEG-PLLA-DA could not polymerize into hydrogels and exhibited a dramatic decrease in autofluorescence, suggesting that hydrolysis of the ester bond affected its autofluorescence. At low pH, PEG-PLLA-DA exhibited higher fluorescence and it was able to form crosslinked hydrogels. The autofluorescence of PEG-PLLA-DA could be exploited to monitor polymer degradation and material structure without the need to introduce exogenous fluorescent probes. The origin of fluorescence is not clear at this point in time but it appears to result from a synergetic effect of both lactate units and diacrylate groups in the PEG-PLLA-DA backbone. The observed autofluorescence of PEG-PLLA-DA persists after reaction of the acrylate groups in the polymerization reaction. This autofluorescence is advantageous because it could assist in the study of polymers used for drug delivery and tissue engineering applications.     

Study of ProtoPorphyrin IX Elimination by Body Excreta: A new Noninvasive Cancer Diagnostic Method?

This paper describes the elimination of porphyrins by feces. It was demonstrated that porphyrin accumulates substantially more in tumors than in normal tissues, and consequently more PPIX reaches the blood of patients and animals with tumors, and then, it needs to be eliminated. The fluorescence of feces revealed that there are large amounts of PPIX in the excreta of animals with cancer comparing with healthy animals. The autofluorescence of feces porphyrin extracted with acetone was analyzed using fluorescence spectroscopy of animals inoculated with DU145 cells into the prostate and healthy animals to monitor the PPIX concentration. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences were observed in autofluorescence intensities measured in the 575–725 nm spectral regions for the studied groups. The results showed a noninvasive, simple, rapid and sensitive method to detect cancer by feces analysis.     

丹酰-L-苯丙氨酸和L-苯丙氨酸对小牛肠碱性磷酸酶抑制作用动力学的比较性研究

利用紫外分光光度法揭示了丹酰-L-苯丙氨酸对小牛肠碱性磷酸酶的选择性抑制作用,并比较性地研究了丹酰-L-苯丙氨酸和L-苯丙氨酸对小牛肠碱性磷酸酶的动力学抑制过程.结果表明在37℃和碱性磷酸酶工作的最佳pH值(pH 10.4)的条件下,丹酰-L-苯丙氨酸与L-苯丙氨酸类似,能够有效抑制小牛肠碱性磷酸酶的活性;利用双倒数曲线拟合,判定丹酰-L-苯丙氨酸与L-苯丙氨酸的抑制类型均为反竞争性抑制,两者的抑制常数Ki均为mmol级.对丹酰-L-苯丙氨酸抑制小牛肠碱性磷酸酶的作用研究,不仪有助于进一步阐明L-苯丙氨酸埘组织特异性碱性磷酸酶的选择性抑制机理,而且丹酰基团的存在也为荧光标定碱性磷酸酶的特效抑制剂提供了可能.     

基于神经网络模式识别的糖尿病无创风险评估方法研究

晚期糖基化终末产物在人体皮肤组织中的浓度与高血糖水平密切相关,且具有自发荧光特性。使用自行研制的光学无创检测装置对人体皮肤组织的自体荧光光谱进行测量,建立神经网络模式识别模型对检测对象患有糖尿病的可能性进行风险评估。利用检测装置获取荧光光谱后对光谱数据进行主成分分析,选取前4个主成分作为光谱的特征,建立一个具有4个输入层节点、6个隐层节点、1个输出节点的神经网络模式识别模型。选取在安徽省立医院测量的487例对象数据训练该模型,以70%数据作为训练集,15%数据作为验证集,15%数据作为测试集。模型可给出测试对象罹患糖尿病的风险,或直接给出是否糖尿病的判断。结果显示该模型的受试者工作特性曲线的线下面积为0.81,标准误差为0.02;以模型输出0.5为分类界限时的敏感性为72.4%,特异性为77.6%,整体准确率为74.9%。本研究首次提出使用皮肤组织自体荧光结合神经网络模式识别模型对糖尿病进行无创风险评估,实验结果表明该方法的筛查效果优于目前常用的空腹静脉血浆血糖值法和糖化血红蛋白法。     

Microfluorescent analysis for bioindication of ozone on unicellular plant systems

Various unicellular model plant systems, on which autofluorescence, fluorescence after histochemical treatment, and growth rates were investigated, have been proposed as bioindicators of ozone. Analysis is performed using fluorescent microscopy in different modifications, including microspectrofluorimetry and laser scanning confocal microscopy. It is found that low ozone doses (0.005 μLL^?1 for 2.5-h exposure or 0.008 μLL^?1 for 4-h exposure) do not affect or stimulate autofluorescence of the samples. In addition, vegetative microspores and pollen retain their ability to germinate in an artificial medium, and their growth is even enhanced. Higher ozone concentrations lead to either a decrease in the emission intensity or a shift of peaks in the fluorescence spectrum. In particular, 16-h exposure of vegetative microspores of horsetail to ozone (total dose 0.032 μLL^?1) leads to occurrence of a new peak in the wavelength range of 515 to 520 nm in their autofluorescence spectrum. Exposure to high (more than 0.1 μLL^?1) doses gives rise to a similar peak in the spectrum of leaf secretory hairs of Raphanus sativus. The spectrum of leaf secretory hairs of Fragaria viridis exhibits a decrease in the emission intensity at wavelengths of 520 to 550 nm. Stress metabolites have been revealed by fluorescence at wavelengths from 460 to 480 nm after specific histochemical reactions for determination of catecholamines or histamine. After exposure in ozone to a total dose of 0.012 μLL^?1, a significant increase in the amount of catecholamines and the histamine content was observed for pollen of Populus balsamifera. Higher concentrations of catecholamines (in comparison with control samples) are found in vegetative microspores of Equisetum arvense and pollen of Corylus avellana and Populus balsamifera after exposure in ozone to high doses (0.032 μLL^?1), whereas a decrease in the catecholamine concentration was observed for pollen of Tulipa hybridum and Dolichothele albescens after this treatment.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术，对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究，尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明，利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团，利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具，对眼底细胞随年龄增长的衰老机理的研究具有重要的意义.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术,对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究,尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明,利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团,利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具,对眼底细胞随年龄增长的衰老机理的研究具有重要的意义. 关键词： 双光子激发荧光 荧光寿命成像 视网膜色素上皮层     

Steady state and time-resolved autofluorescence studies of human colonic tissues

Steady state and time-resolved autofluorescence spectroscopies are employed to study the autofluorescence characteristics of human colonic tissues in vitro. The excitation wavelength varies from 260 to 540 nm, and the corresponding fluorescence emission spectra are acquired from 280 to 800 nm. Significant difference in fluorescence intensity of excitation-emission matrices (EEMs) is observed between normal and tumor colonic tissues. Compared with normal colonic tissue, low nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD), and high amino acids and protoporphyrin Ⅸ (PpⅨ) fluorescences characterize high-grade malignant tissue. Moreover, the autofluorescence lifetimes of normal and carcinomatous colonic tissues at 635 nm under 397-nm excitation are about 4.32±0.12 and 18.45±0.05 ns, respectively. The high accumulation of endogenous PpⅨ in colonic cancers is demonstrated in both steady state and time-resolved autofluorescence spectroscopies.     

基于口腔自体荧光效应和深度学习的牙菌斑的分割及量化

牙菌斑是牙齿表面一层难以观测的生物膜,是导致龋齿、牙龈炎等一系列疾病的直接诱因.牙菌斑的早期定量化无损检测具有重要的临床意义.短波长光激发下,牙菌斑的细菌及其代谢产物可以产生自体荧光.基于前期成像系统的基础上,采集大量牙菌斑在405nm蓝光激发下产生的红色荧光;牙菌斑越成熟红色荧光强度越高;采用改进的U-net网络对该...     

Measurement Conditions for Flow Cytometry Analyses of Cell Lines from Urological Carcinomas

Prerequisites for successful flow cytometry investigations are specific antibodies labeled with appropriate fluorochromes and negligible autofluorescence of the untreated cells at the wavelength of interest. The aim of this study was (a) to characterize frequently used urological carcinoma cell lines with regard to their autofluorescence properties, (b) to demonstrate the autofluorescence as a serious interfering factor on FACS analysis of urological carcinoma cell lines and (c) to suggest an alternative to avoid interfering autofluorescence. Twenty-one cell lines originating from prostate carcinoma, renal cell carcinoma and bladder cancer were included in this study. The various cell lines were read on a flow cytometer in comparison to human erythrocytes as cells with low fluorescence intensity. Urological cell lines show a high autofluorescence when flow cytometry analyses are performed at the frequently used excitation wavelengths at 405 and 488 nm. At excitation wavelength of 633 nm, this problem was reduced and most of the cell lines (14/21) were without autofluorescence at the emission wavelength of 785 nm. In addition, with a spectrofluorometer three exemplary cell lysates were investigated. The above observations were confirmed. The dye APC-Cy7 is one suitable fluorochrome for successful investigation under these measurement conditions.     

Study of Blood Porphyrin Spectral Profile for Diagnosis of Tumor Progression

Renal cell carcinoma (RCC) accounts for approximately 3% of new cancer incidence and mortality in the United States. Unfortunately many RCC masses remain asymptomatic and nonpalpable until they are advanced. Diagnosis and localization of early carcinoma play an important role in the prevention and curative treatment of RCC. The autofluorescence of blood porphyrin of healthy and tumor induced in male SCID mice was analyzed using fluorescence and excitation spectroscopy. A significant contrast between normal and tumor blood could be established. Blood porphyrin fluorophore showed enhanced fluorescence band (around 630 nm) in function of the tumor growth. This indicates that either the autofluorescence intensity of the blood fluorescence may provide a good parameter for the “first approximation” characterization of the tumor stage.     

强荧光背景环境中荧光涨落谱--蒙特卡罗模拟研究

荧光涨落谱方法(Fluorescence Fluctuation Spectroscopy)通过分析微小探测区域内的荧光涨落信号,获得粒子亮度、扩散系数以及溶液浓度等信息。利用Monte Carlo模拟方法,研究了溶液中自荧光背景和系统噪声对荧光涨落谱的影响。结果表明,利用双组分光子计数统计方法,可以有效去除低亮度、高浓度背景组分自荧光和均匀分布系统噪声产生的影响。本结果为利用荧光涨落谱方法测量细胞体系复杂环境中的蛋白质相互作用提供帮助。     

Optical processing of light-induced autofluorescence for characterization of tissue pathology

We describe an optical processing method for characterizing tissue pathology that is based on principal-component analysis of light-induced autofluorescence. A set of optical spectral filters, which are related to the principal-component loading vectors, is designed to process the autofluorescence signal optically and to generate principal-component scores from the autofluorescence spectra. The scores are then correlated with the tissue pathology. An optical processing system is designed that uses the in vivo fluorescence spectra recorded from nasopharyngeal tissues. We demonstrate that the system can differentiate nasopharyngeal carcinoma from normal tissue with a high degree of sensitivity and specificity and that the optical filters used in the system can be manufactured.