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Global analysis of fluorescence lifetime imaging microscopy data

BIOPHYSICAL JOURNAL 2000年第4期78卷 2127-2137页

作者： Verveer, PJ Squire, A Bastiaens, PIH Imperial Canc Res Fund Cell Biophys Lab London WC2A 3PX England

Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.

关键词：算法细菌蛋白质类/代谢长尾猴黑数据说明统计荧光染料绿色荧光蛋白质类 HeLa细胞发光蛋白质类/代谢显微镜检查荧光/方法显微镜检查荧光/统计学和数值数据 Vero细胞动物人类

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Incidence of chromosomes 1 and 17 aneusomy in breast cancer and adjacent tissue: An interphase cytogenetic study

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JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS 2000年第5期190卷 530-539页

作者： Botti, C Pescatore, B Mottolese, M Sciarretta, F Greco, C Di Filippo, F Gandolfo, GM Cavaliere, F Bovani, R Varanese, A Cianciulli, AM Regina Elena Inst Canc Res Div Surg Oncol Dept Surg Oncol I-00161 Rome Italy Regina Elena Inst Canc Res Dept Clin Pathol I-00161 Rome Italy Regina Elena Inst Canc Res Dept Pathol I-00161 Rome Italy

Background: Characterization of the biopathologic events underlying the early steps of breast carcinogenesis may have a dramatic impact on reducing breast cancer mortality. Genes involved in breast tumorigenesis are localized on chromosomes 1 and 17, and numeric aberrations of these chromosomes have been correlated with breast cancer tumorigenesis and progression. According to the field cancerization hypothesis, specific chromosome aberrations may be present in breast cancer and in normal-appearing adjacent tissue. The latter changes reflect the genomic damage that follows longterm carcinogenic exposure and precede the morphologically detectable neoplastic transformation. We hypothesize that detection of these aberrations in benign breast epithelium may provide a tool for molecular risk assessment. Study Design: Using fluorescence in situ hybridization with centromere-specific probes, we determined the status of chromosomes 1 and 17 in fresh imprints of 28 samples of primary tumors and 54 samples of their surrounding uninvolved parenchyma taken from patients undergoing operations for breast carcinoma. Ten contralateral breast biopsy specimens collected from patients with previous breast carcinoma were also evaluated as a surrogate of a high-risk group to rule out the hypothesis that chromosomal aneusomy in tumor-adjacent tissue could be related to a paracrine effect of the primary tumor Ten samples of benign breast tissue taken from patients at low risk were used as controls to define tolerance limits for aneusomy definition. Results: Using threshold values of 40% of signal loss and 13% of signal gain to define chromosome aneusomy tie, mean + 3 SDs of the control group signals), we found the following: 1) almost all primary breast tumors were aneusomic for chromosomes 1 and 17;2) primary breast tumor and adjacent uninvolved parenchyma shared the same pattern of chromosomes 1 and 17 aneusomy in 66.7% of patients;and 3) chromosomes 1 and 17 aneusomies in contralateral

关键词：非整倍性活组织检查乳房/病理学乳腺肿瘤/遗传学乳腺肿瘤/病理学染色体人 1对/遗传学染色体人 17对/遗传学原位杂交荧光/统计学和数值数据发病率分裂间期/遗传学危险性评估成年人女(雌)性人类中年人

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Two-dimensional fluorescence intensity distribution analysis:: Theory and applications

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BIOPHYSICAL JOURNAL 2000年第4期78卷 1703-1713页

作者： Kask, P Palo, K Fay, N Brand, L Mets, Ü Ullmann, D Jungmann, J Pschorr, J Gall, K EVOTEC BioSyst AG D-22522 Hamburg Germany Inst Expt Biol EE-76902 Harku Estonia

A method of sample analysis is presented which is based on fitting a joint distribution of photon count numbers. In experiments, fluorescence from a microscopic volume containing a fluctuating number of molecules is monitored by two detectors, using a confocal microscope. The two detectors may have different polarizational or spectral responses. Concentrations of fluorescent species together with two specific brightness Values per species are determined. The two-dimensional fluorescence intensity distribution analysis (2D-FIDA), if used with a polarization cube, is a tool that is able to distinguish fluorescent species with different specific polarization ratios. As an example of polarization studies by 2D-FIDA, binding of 5'-(6-carboxytetramethylrhodamine) (TAMRA)-labeled theophylline to an anti-theophylline antibody has been studied. Alternatively, if two-color equipment is used, 2D-FIDA can determine concentrations and specific brightness values of fluorescent species corresponding to individual labels alone and their complex. As an example of two-color 2D-FIDA, binding of TAMRA-labeled somatostatin-14 to the human type-2 high-affinity somatostatin receptors present in stained vesicles has been studied. The presented method is unusually accurate among fluorescence fluctuation methods. It is well suited for monitoring a variety of molecular interactions, including receptors and ligands or antibodies and antigens.

关键词：算法抗原抗体反应生物物理学评价研究(主题) 荧光偏振/仪器和设备荧光偏振/方法荧光偏振/统计学和数值数据荧光染料显微镜检查共焦/仪器和设备显微镜检查共焦/方法显微镜检查共焦/统计学和数值数据显微镜检查荧光/仪器和设备显微镜检查荧光/方法显微镜检查荧光/统计学和数值数据模型理论光子受体生长抑素/代谢若丹明类生长抑素/代谢茶碱/分析茶碱/免疫学人类

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Determination of D-sorbitol in human erythrocytes by an enzymatic fluorometric method with an improved deproteinization procedure

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ANNALS OF CLINICAL BIOCHEMISTRY 2001年第6期38卷 701-707页

作者： Umeda, M Otsuka, Y Ii, T Matsuura, T Shibati, H Ota, H Sakurabayashi, I Nissui Pharmaceut Co Ltd Div Res Yuki Ibaraki 307 Japan Jichi Med Sch Omiya Med Ctr Dept Clin Labs Omiya Saitama 330 Japan

We developed an improved enzymatic assay of D-sorbitol in human erythrocytes by employing highly specific D-sorbitol dehydrogenase from Pseudomonas sp. (EC 1.1.1.14) and replacing perchloric acid (HClO4) and potassium carbonate (K2CO3), generally used for deproteinization, with sodium hydroxide (NaOH) and zinc sulphate (ZnSO4). In this assay, erythrocytes were separated from plasma by centrifugation and washed once with physiological saline. Subsequently, the erythrocytes were lysed with distilled water and proteins precipitated with NaOH and ZnSO4. After centrifugation, the resulting colourless supernatant was mixed with a glycine buffer (pH 9.0) containing NAD(+) and D-sorbitol dehydrogenase. After incubation for 30 min at 37 degreesC, the NADH produced was measured fluorimetrically. The fluorescence intensities were corrected for sample blanks, and the values of D-sorbitol were normalized for haemoglobin content. The method had an analytical range of 1-180 mu mol/L. The intra- and inter-assay precisions were < 3.3% and < 5.8%, respectively. The detection limit was 0.65 mu mol/ L. In terms of the linearity, precision and sensitivity, the improved method using NaOH and ZnSO4 was superior to the conventional method using HClO4 and K2CO3.

关键词：血液化学分析/方法血液化学分析/统计学和数值数据血蛋白质类/分离和提纯红细胞/化学氢离子浓度 L-艾杜糖醇-2-脱氢酶 NAD 敏感性与特异性山梨醇/血液光谱法荧光/方法光谱法荧光/统计学和数值数据人类

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A theoretical investigation of single-molecule fluorescence detection on thin metallic layers

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BIOPHYSICAL JOURNAL 2000年第4期78卷 2151-2158页

作者： Enderlein, J Univ Regensburg Inst Analyt Chem Chemo & Biosensors D-93040 Regensburg Germany

In the present paper, the excitation and detection of single-molecule fluorescence over thin metallic films is studied theoretically within the framework of classical electrodynamics. The model takes into account the specific conditions of surface plasmon-assisted optical excitation, fluorescence quenching by the metal film, and detection geometry. Extensive numerical results are presented for gold, silver, and aluminum films, showing the detectable fluorescence intensities and their dependence on film thickness and the fluorescent molecule's position under optimal excitation conditions.

关键词：铝生物物理学电化学金显微镜检查荧光/方法显微镜检查荧光/统计学和数值数据模型理论银表面等离激元

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