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通过共聚焦激光扫描显微镜进行定量单分子成像。

Quantitative single-molecule imaging by confocal laser scanning microscopy.

作者信息

Vukojevic Vladana, Heidkamp Marcus, Ming Yu, Johansson Björn, Terenius Lars, Rigler Rudolf

机构信息

Department of Clinical Neuroscience, Karolinska Institutet, 17176 Stockholm, Sweden.

出版信息

Proc Natl Acad Sci U S A. 2008 Nov 25;105(47):18176-81. doi: 10.1073/pnas.0809250105. Epub 2008 Nov 14.

Abstract

A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed.

摘要

提出了一种通过共聚焦激光扫描显微镜(CLSM)进行定量单分子成像的新方法。它依靠荧光强度分布来分析数字成像捕获的分子出现统计数据,并且无需借助时间或空间自相关分析就能直接确定荧光分子的数量及其扩散速率。通过使用快速扫描和雪崩光电二极管探测器记录荧光分子的数字图像。通过这种方式,显著提高了信噪比,从而实现了CLSM的直接定量成像。通过在溶液和活细胞中使用荧光染料标准溶液、荧光标记的DNA分子、量子点和增强型绿色荧光蛋白,证明了所提出方法的潜力。该方法通过荧光相关光谱法进行了验证。讨论了其在生物应用中的相关性,特别是在活细胞成像方面的相关性。

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