Fluorescence Photobleaching and Fluorescence Correlation Spectroscopy: Two Complementary Technologies To Study Molecular Dynamics in Living Cells

  • Malte Wachsmuth
  • Klaus Weisshart
Part of the Principles and Practice book series (PRINCIPLES)

Fluorescence recovery or redistribution after photobleaching (FRAP) and fluorescence correlation or fluctuation spectroscopy (FCS) are probably the most widely used techniques employed to study the transport and diffusion as well as the interaction and immobilisation of biological molecules inside the cellular environment. This has been promoted by the emergence of fluorescent proteins for in vivo labelling and the development of confocal laser scanning microscopes that also allow for photobleaching and fluctuation spectroscopy experiments. FRAP represents a family of methods which are all based on the photoinduced bleaching (or activation) of marker molecules in selected areas of a cell followed by the relaxation back to equilibrium. FCS stands for another and complementary set of relaxation methods which are based on the observation and analysis of thermal fluctuations of sparse labelled molecules in a microscopic observation volume. Being conceptually different, these techniques taken together and combined with confocal imaging give access to a wide time and concentration range and can yield qualitatively and quantitatively biochemical and biophysical data such as concentrations, reaction rates, free and bound fractions, or diffusion coefficients. We present aspects of the biological and physical background, outline typical fields of applications, and give some guidance on how to carry out FRAP, FCS, and continuous photobleaching experiments with an emphasis on practical aspects and pitfalls.


Green Fluorescent Protein Fluorescence Correlation Spectroscopy Confocal Volume Pinhole Size Anomalous Subdiffusion 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2007

Authors and Affiliations

  • Malte Wachsmuth
    • 1
  • Klaus Weisshart
    • 2
  1. 1.Cell Biophysics GroupInstitut Pasteur KoreaSeongbukgu, SeoulRepublic of Korea
  2. 2.Carl Zeiss MicroImaging GmbHCarl-Zeiss-Promenade 10JenaGermany

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