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Fluorescence Correlation Spectroscopy: Principles and Developments

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Supramolecular Structure and Function 10

Abstract

Twenty years ago, fluorescence measurements at low concentrations were difficult due to the weak fluorescence signal and intrinsic fluctuations of the sample. With the development of FCS and its implementation on a confocal microscope, it is possible to use the inherent fluctuations to gain information over the concentration, molecular brightness, microscopic rate constants for reactions and mobility of the measured sample. In recent years, there has been a strong increase in the development and application of fluctuation methods. With pulsed interleaved excitation, stoichiometry information can be obtained and spectral cross-talk can be eliminated from FCCS experiments. An elegant implementation of two-focus FCS has also been introduced to allow absolute measurements of diffusion coefficient without precise knowledge of the psf of the microscope and is less sensitive to the laser excitation intensity and saturation effects. Scanning methods such as Scanning FCS and RICS increase the effective volume, which is advantageous for live-cell measurements where diffusion is slow and photobleaching is a problem. In this article, describe the basics of FCS and its limitations as well as a short discussion of a handful of emerging techniques. There are still many other equally interesting applications of fluorescence fluctuation spectroscopy that we have not been able to touch upon. And, if the past is any indication of the future, there will be a number of novel fluorescence fluctuation spectroscopy methods emerging in the near future.

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Notes

  1. 1.

    It is also possible to perform FCS using Total Internal Reflection Excitation, but a description of this method is beyond the scope of this publication. For details see Thompson et al. (1981).

  2. 2.

    For simplicity, we assume that the quantum yields of the green and red fluorophores are identical in the single and double-labeled complexes. The more general equations can be found in Müller et al. Biophys J 89:3508.

  3. 3.

    Polarized detection alone would not be sufficient to separate the two volumes as the detected fluorescence is strongly depolarized due to rotation of the fluorophores on the time scale of the fluorescence lifetime.

Abbreviations

2fFCS:

Two-focus fluorescence correlation spectroscopy

ACF:

Autocorrelation function

ALEX:

Alternating laser excitation

CCF:

Cross-correlation function

ccRISC:

Cross-correlation raster image correlation spectroscopy

FCS:

Fluorescence correlation spectroscopy

FCCS:

Fluorescence cross-correlation spectroscopy

FRET:

Förster resonance energy transfer

PIE:

Pulsed interleaved excitation

RISC:

Raster image correlation spectroscopy

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Authors and Affiliations

  1. Department for Chemistry and Biochemistry, Center for Nanoscience (CeNS) and Munich Center for Integrated Protein Science (CiPSM), Ludwig-Maximilians-Universität München, 81377, Munich, Germany

    Sergey Ivanchenko

  2. Department for Chemistry, Center for Nanoscience (CeNS) and Munich Center for Integrated Protein Science (CiPSM), Ludwig-Maximilians-Universität München, 81377, Munich, Germany

    Don C. Lamb

  3. Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, USA

    Don C. Lamb

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  1. Sergey Ivanchenko
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  2. Don C. Lamb
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Correspondence to Don C. Lamb .

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  1. , School of Medicine, University of Zagreb, Ŝalata 3, Zagreb, 10000, Croatia

    Jasminka Brnjas-Kraljević

  2. Ruder Boskovic Institute, Bijenicka 54, Zagreb, 10000, Croatia

    Greta Pifat-Mrzljak

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Ivanchenko, S., Lamb, D.C. (2011). Fluorescence Correlation Spectroscopy: Principles and Developments. In: Brnjas-Kraljević, J., Pifat-Mrzljak, G. (eds) Supramolecular Structure and Function 10. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-0893-8_1

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