Abstract
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.
Highlights
Raster image correlation spectroscopy (RICS) is a noninvasive image analysis technique to measure the diffusion coefficient and concentration of fluorescently labeled proteins in cells[1,2]
Developed by Digman et al.[1], RICS is accessible to many researchers because it can be performed using a commercially available laser scanning microscope
Experiments described here—In this article, we describe how to perform RICS calibration and measurements with the commercially available Olympus FluoView FV1000 confocal laser scanning microscope using Olympus FluoView software to acquire data and SimFCS software to analyze the results
Summary
Raster image correlation spectroscopy (RICS) is a noninvasive image analysis technique to measure the diffusion coefficient and concentration of fluorescently labeled proteins in cells[1,2]. The horizontal width of the image correlation is equal to the number of pixels that need to be skipped when fitting RICS data. Equation 3 describes the maximum value for τp Another important consideration in RICS analysis is the number of frames to be used for moving-average background subtraction.
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