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Fluorescence correlation spectroscopy in living cells

Nature Methods volume 4, pages 963–973 (2007)Cite this article

Fluorescence correlation spectroscopy (FCS) is an ideal analytical tool for studying concentrations, propagation, interactions and internal dynamics of molecules at nanomolar concentrations in living cells1,2,3,4. FCS analyzes minute fluorescence-intensity fluctuations about the equilibrium of a small ensemble (<103) of molecules. These fluctuations act like a 'fingerprint' of a molecular species detected when entering and leaving a femtoliter-sized optically defined observation volume created by a focused laser beam. In FCS the fluorescence fluctuations are recorded as a function of time and then statistically analyzed by autocorrelation analysis. The resulting autocorrelation curve yields a measure of self-similarity of the system after a certain time delay, and its amplitude describes the normalized variance of the fluorescence fluctuations. By fitting the curves to an appropriate physical model, this method provides precise information about a multitude of measurement parameters, including diffusion coefficients, local concentration, states of aggregation and molecular interactions. FCS operates in real time with diffraction-limited spatial and sub-microsecond temporal resolution. Assessing diverse molecular dynamics within the living cell is a challenge well met by FCS because of its single-molecule sensitivity and high dynamic resolution3,4. For these same reasons, however, intracellular FCS measurements also harbor the large risk of collecting artifacts and thus producing erroneous data. Here we provide a step-by-step guide to the application of FCS to cellular systems, including methods for minimizing artifacts, optimizing measurement conditions and obtaining parameter values in the face of diverse and complex conditions of the living cell. A discussion of advantages and disadvantages of one-photon versus two-photon excitation for FCS is available in Supplementary Methods online.

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Figure 1: FCS setup.
Figure 2: Minimizing artifacts in intracellular FCS measurements.
Figure 3: FCS measurements in cells.

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Acknowledgements

We thank K. Bacia for critical reading of the manuscript and valuable comments, and E. Haustein, K. Bacia, M.N. Waxham, D.R. Larson and W.R. Zipfel for helpful and constructive discussions. This work was supported by grants of the Fulbright Kommission to S.A.K., the German Ministry for Education and Research to P.S. and Human Frontiers to P.S.

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Author notes

  1. Sally A Kim & Katrin G Heinze

    Present address: Present addresses: Division of Biology, 114-96, California Institute of Technology, Pasadena, California 91125, USA (S.A.K.) and Research Institute for Molecular Pathology, Dr. Bohr Gasse 7, 1030 Vienna, Austria (K.G.H.).,

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  1. Institute of Biophysics, Dresden University of Technology, Tatzberg 47-51, Dresden, D-01307, Germany

    Sally A Kim, Katrin G Heinze & Petra Schwille

Authors
  1. Sally A Kim
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  2. Katrin G Heinze
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  3. Petra Schwille
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Correspondence to Petra Schwille.

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Supplementary Table and Methods

Supplementary Table 1 and Supplementary Methods (PDF 232 kb)

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Kim, S., Heinze, K. & Schwille, P. Fluorescence correlation spectroscopy in living cells. Nat Methods 4, 963–973 (2007). https://doi.org/10.1038/nmeth1104

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