Photobleaching Measurements of Diffusion in Cell Membranes and Aqueous Cell Compartments

György Lustyik1

1 University of Pécs, Faculty of Medicine, Pécs, Hungary
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.12
DOI:  10.1002/0471142956.cy0212s16
Online Posting Date:  May, 2001
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Abstract

This commentary unit discusses in great detail the theoretical nature of fluorescence recovery after photobleaching (FRAP). This information is crucial to an understanding of how and why FRAP works in a cell system. Further, understanding how to interpret the data sets requires a sound knowledge of the processes involved. Of primary importance are the nature of membrane diffusion and the nature of the multiple compartments into which fluorescent dyes can enter. The unit provides a complete discussion of all aspects of FRAP from the perspective of cellular measurements.

     
 
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Table of Contents

  • Diffusion in the Membrane
  • Linearization Methods
  • Multi‐Component FRAP Models
  • Anomalous Diffusion
  • Diffusion in Aqueous Compartments
  • Single Molecule Detection
  • Practical Guidelines
  • Conclusions
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

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Figures

  •   FigureFigure 2.12.1 Schematic diagram of a conventional FRAP instrument. The basic unit of the optical system is a fluorescence microscope. A laser beam is used for both fluorescence excitation and photobleaching. The attenuated excitation beam and the high‐intensity main beam illuminate the very same region of the specimen. Attenuation can be achieved with neutral‐density filters or with a beam splitter. On the figure, the attenuated beam is achieved by 4‐fold reflection on a pair of precisely aligned optical flats. The shutter excludes the main beam when it is closed, and allows the beam to pass when it is open. OF, optical filter; PMT, photomultiplier.
  •   FigureFigure 2.12.2 A typical recording of a FRAP measurement. The fluorescence intensity ( Fo) measured in a small region of the sample (typically 3 to 4 µm in diameter) is proportional to the average concentration of fluorochrome in that region. At t = 0, the small region is illuminated with a strong light pulse (bleaching). The fluorochrome in the region undergoes an irreversible, light‐induced photo‐decomposition and the post‐bleaching fluorescence intensity drops to a lower level ( F(0)). The fluorescence intensity shows a recovery because intact fluorochrome molecules diffuse into the bleached area due to the Brownian motion. The F(∞) intensity of full recovery at long time is lower than Fo if the fluorochrome‐labeled diffusing compound is partly immobile.

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Literature Cited

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