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Fluorescence correlation spectroscopy and fluorescence cross‐correlation spectroscopy

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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. This article is categorized under: Laboratory Methods and Technologies > Proteomics Methods

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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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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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(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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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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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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