Fluorescence correlation spectroscopy and fluorescence cross‐correlation spectroscopy
Focus Article
Michelle A. Digman, Enrico Gratton
Published Online: Apr 29 2009
DOI: 10.1002/wsbm.5
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Abstract This article focuses on methods based on fluctuation correlation spectroscopy to determine the formation of protein complexes
in living cells. We present the principles of the fluctuation method applied to cells. We discuss the novelty and the promises
of this approach. The emphasis is in the discussion of the underlying statistical assumptions of the image correlation spectroscopy
analysis rather than in reviewing applications of the method. Although one example of the application of the fluctuation method
is given, this article also contains simulations that are better suited to illustrate and support the basic assumptions of
the method. Copyright © 2009 John Wiley & Sons, Inc.
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Figure 1. Image correlation spectroscopy (ICS): simulations of aggregates of different sizes. (a) 50 nm, (b) 100 nm, (c) 400 nm, (d) 800 nm, (e) 1600 nm, and (f) combination of 50 nm and 1600 nm particles. For the simulation, the point spread function (PSF) was 300 nm, pixel size 50 nm, the image was 256 × 256 pixels and 100 particles were used.
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Figure 2. Simulations: (a) G(0,0) is proportional to the inverse of the number of particles N. (b) Fraction of cross-correlation as a function of the fraction of double-labeled particles at 10% bleedthrough.
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Figure 3. Differences between (a) raster scan and (b) camera snapshot acquisition mode. (c) and (d) are the correlation functions for (a) and (b) images. Parameters for the simulation were identical to these described for Figure 1 . The diffusion coefficient of the particles was 10 µm2 / s and the pixel dwell time was 15.2 µs.
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Figure 4. (a) Vinculin-EGFP and (b) Paxillin-mCherry in MEF cell. (c) and (d) RICS autocorrelation functions and (e) Cross-correlation. There is no appreciable cross-correlation. The small amplitude is the bleedthrough.
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Figure 5. Vinculin-EGFP and paxillin-mCherry in MEF cells. The entire adhesion is moving in macroscopic concerted apparent motion. The spatiotemporal image correlation spectroscopy (STICS) correlation functions are at delays (a) 0 s, (b) 4 s, (C) 8 s, (D) 12 s, (E) 16 s and (F) 20 s.
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References 1 Clegg RM. FRET tells us about proximities, distances, orientations and dynamic properties J Biotechnol 2002 82:177-179 et al. Measuring fast dynamics in solutions and cells with a laser scanning microscope Biophys J 2005 89:1317-1327 et al. Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure Biophys J 2005 88:L33-L36 et al. Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope J Microsc 2008 229:78-91
Resources
Further Readings
Brown, C.M. et al. Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J Microsc 229, 78-91 (2008).
Digman, M.A., Brown, C.M., Horwitz, A.R., Mantulin, W.W. & Gratton, E. Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy. Biophys J 94, 2819-2831 (2008).
Magde, D., Elson, E.L. & Webb, W.W. Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13, 29-61 (1974).
Elson, E.L. & Webb, W.W. Concentration correlation spectroscopy: a new biophysical probe based on occupation number fluctuations. Annu Rev Biophys Bioeng 4, 311-334 (1975).
Elson, E.L. Fluorescence correlation spectroscopy measures molecular transport in cells. Traffic 2, 789-796 (2001).
Elson, E.L. Quick tour of fluorescence correlation spectroscopy from its inception. J Biomed Opt 9, 857-864 (2004).
