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Quantifying intracellular dynamics using fluorescence fluctuation spectroscopy

  • Special Issue: New/Emerging Techniques in Biological Microscopy
  • Published:
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Abstract

Originally developed for the field of physical chemistry, fluorescence fluctuation spectroscopy (FFS) has evolved to a family of methods to quantify concentrations, diffusion rates and interactions of fluorescently labelled molecules. The possibility to measure at the nanomolar concentration level and to combine these techniques with microscopy allow to study biological processes with high sensitivity in the living cell. In this review, the basic principles, challenges and recent developments of the most common FFS methods are being discussed and illustrated by intracellular applications.

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Abbreviations

CFP:

Cyan fluorescent protein

CMOS:

Complementary metal oxide semiconductor

EM-CCD:

Electron-multiplying charge-coupled device

FCCS:

Fluorescence cross-correlation spectroscopy

FCS:

Fluorescence correlation spectroscopy

FFS:

Fluorescence fluctuation spectroscopy

FIDA:

Fluorescence intensity distribution analysis

FP:

Fluorescent protein

FRAP:

Fluorescence recovery after photobleaching

FRET:

Förster resonance energy transfer

GFP:

Green fluorescent protein

HD:

Hybrid detector

ICS:

Image correlation spectroscopy

MAPK:

Mitogen-activated protein kinase

N&B:

Number and brightness analysis

PCH:

Photon-counting histogram

RICS:

Raster image correlation spectroscopy

SPIDA:

Spatial intensity distribution analysis

SPIM:

Selective plane illumination microscopy

STED:

Stimulated emission depletion

STICS:

Spatio-temporal image correlation spectroscopy

TIRF:

Total internal reflection fluorescence

YFP:

Yellow fluorescent protein

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Acknowledgments

The author would like to thank the Netherlands Organisation for Scientific Research (NWO) for supporting research via Echo & Middelgroot investment grants.

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  1. Section of Molecular Cytology, van Leeuwenhoek Centre for Advanced Microscopy (LCAM), Swammerdam Institute for Life Sciences (SILS), University of Amsterdam, Sciencepark 904, 1098 XH, Amsterdam, The Netherlands

    Mark A. Hink

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Correspondence to Mark A. Hink.

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Handling Editor: J. W. Borst

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Hink, M.A. Quantifying intracellular dynamics using fluorescence fluctuation spectroscopy. Protoplasma 251, 307–316 (2014). https://doi.org/10.1007/s00709-013-0602-z

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