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Molecular diffusion and binding analyzed with FRAP

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

Intracellular molecular transport and localization are crucial for cells (plant cells as much as mammalian cells) to proliferate and to adapt to diverse environmental conditions. Here, some aspects of the microscopy-based method of fluorescence recovery after photobleaching (FRAP) are introduced. In the course of the last years, this has become a very powerful tool to study dynamic processes in living cells and tissue, and it is expected to experience further increasing demand because quantitative information on biological systems becomes more and more important. This review introduces the FRAP methodology, including some theoretical background, describes challenges and pitfalls, and presents some recent advanced applications.

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Abbreviations

CLSM:

Confocal laser scanning microscopy

CP:

Continuous fluorescence photobleaching

ER:

Endoplasmatic reticulum

FCS:

Fluorescence correlation spectroscopy

FM:

Fluorescence microphotolysis

FPR:

Fluorescence photobleaching recovery

FRAP:

Fluorescence recovery/redistribution after photobleaching

GFP:

Green fluorescent protein

GR:

Glucocorticoid receptor

HP1:

Heterochromatin protein 1

mRNP:

mRNA–protein complex

MSD:

Mean squared displacement

NA:

Numerical aperture

PSF:

Point-spread function

RICS:

Raster image correlation spectroscopy

ROI:

Region of interest

SD:

Spinning disk

TIRF:

Total internal reflection fluorescence

YFP:

Yellow fluorescent protein

3PEA:

Pixel-wise photobleaching profile evolution analysis

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Acknowledgments

This work was supported by EMBL research funding.

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The author declares that he has no conflict of interest.

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  1. Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany

    Malte Wachsmuth

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  1. Malte Wachsmuth
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Correspondence to Malte Wachsmuth.

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Handling Editor: J. W. Borst

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Wachsmuth, M. Molecular diffusion and binding analyzed with FRAP. Protoplasma 251, 373–382 (2014). https://doi.org/10.1007/s00709-013-0604-x

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  • DOI: https://doi.org/10.1007/s00709-013-0604-x

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