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Raster image correlation spectroscopy in live cells


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.

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Figure 1: LightPath & Dyes window in Olympus FluoView 1.7a.
Figure 2: The Acquisition Settings window in Olympus FluoView 1.7a.
Figure 3: The Image Acquisition Control window in Olympus FluoView 1.7a.
Figure 4: LUT window in Olympus FluoView.
Figure 5: Export files window in Olympus FluoView 1.7a.
Figure 6: SimFCS RICS analysis screen.
Figure 7: SimFCS fitting window.
Figure 8: Cursor analysis in the SimFCS RICS.
Figure 9: SimFCS pfitform Window.
Figure 10: Erroneous RICS results.
Figure 11: Typical results for RICS calibration measurement of EGFP in solution.
Figure 12: RICS analysis of cytosolic EGFP in a CHO-K1 cell.
Figure 13: RICS analysis of EGFP-paxillin in a CHO-K1 cell.

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This research was supported by the National Institutes of Health (PHS 5 P41-RR003155, U54 GM064346 Cell Migration Consortium and P50-GM076516 grants).

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Authors and Affiliations



M.J.R. was primarily responsible for preparing this article. M.J.R. and J.M.S. conducted the experiments presented here. M.A.D. and E.G., along with others, primarily developed the RICS technique. They provided technical guidance and expertise.

Corresponding author

Correspondence to Enrico Gratton.

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The authors declare no competing financial interests.

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Rossow, M., Sasaki, J., Digman, M. et al. Raster image correlation spectroscopy in live cells. Nat Protoc 5, 1761–1774 (2010).

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