Department of Biomedical Engineering, University of California Irvine, Irvine, California, USA.
Nat Protoc. 2010 Nov;5(11):1761-74. doi: 10.1038/nprot.2010.122. Epub 2010 Oct 14.
Raster image correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in a live cell, including concentration of molecules and diffusion coefficients of molecules; in addition, by measuring changes in diffusion coefficients, RICS can indirectly detect binding. Any specimen containing fluorophores that can be imaged with a laser scanning microscope can be analyzed using RICS. There are other techniques to measure diffusion coefficients and binding; however, RICS fills a unique niche. It provides spatial information and can be performed in live cells using a conventional confocal microscope. It can measure a range of diffusion coefficients that is not accessible with any other single optical correlation-based technique. In this article we describe a protocol to obtain raster scanned images with an Olympus FluoView FV1000 confocal laser scanning microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Each RICS measurement takes several minutes. The entire procedure can be completed in ∼2 h. This procedure includes focal volume calibration using a solution of fluorophores with a known diffusion coefficient and measurement of the diffusion coefficients of cytosolic enhanced green fluorescent protein (EGFP) and EGFP-paxillin.
光栅扫描相关光谱学（RICS）是一种非侵入性技术，用于检测和量化活细胞中的事件，包括分子浓度和分子扩散系数；此外，通过测量扩散系数的变化，RICS 可以间接检测结合。任何含有可以用激光扫描显微镜成像的荧光团的标本都可以用 RICS 进行分析。还有其他技术可以测量扩散系数和结合；然而，RICS 填补了一个独特的空白。它提供空间信息，并且可以在使用传统共焦显微镜的活细胞中进行。它可以测量任何其他单一基于光相关的技术都无法测量的扩散系数范围。在本文中，我们描述了一种使用 Olympus FluoView FV1000 共焦激光扫描显微镜获得光栅扫描图像的方案，使用 Olympus FluoView 软件获取数据，使用 SimFCS 软件进行 RICS 分析。每个 RICS 测量需要几分钟。整个过程可以在 2 小时内完成。该程序包括使用具有已知扩散系数的荧光团溶液进行焦体积校准，以及测量胞质增强型绿色荧光蛋白（EGFP）和 EGFP-桩蛋白的扩散系数。
