Unit

UNIT 21.2 Fluorescence Localization After Photobleaching (FLAP)

  1. Graham A. Dunn1,
  2. Mark R. Holt1,
  3. Daniel Y. H. Soong1,
  4. Colin Gray2,
  5. Daniel Zicha2

Published Online: 1 OCT 2004

DOI: 10.1002/0471143030.cb2102s24

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Dunn, G. A., Holt, M. R., Soong, D. Y. H., Gray, C. and Zicha, D. 2004. Fluorescence Localization After Photobleaching (FLAP). Current Protocols in Cell Biology. 24:21.2:21.2.1–21.2.16.

Author Information

  1. 1

    The Randall Division, King's College London, London, United Kingdom

  2. 2

    Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, London, United Kingdom

Publication History

  1. Published Online: 1 OCT 2004
  2. Published Print: SEP 2004

Abstract

Fluorescence localization after photobleaching is a new method for localized photolabeling and subsequent tracking of specific molecules within living cells. The molecular species to be located carries two different fluorophores that can be imaged independently but simultaneously by fluorescence microscopy. For the method to work, these two fluorophores should be accurately colocalized throughout the cell so that their images are closely matched. One of the fluorophores (the target fluorophore) is then rapidly photobleached at a chosen location. The unbleached (reference) fluorophore remains colocalized with the target fluorophore; thus, the subsequent fate of the photobleached molecules can be revealed by processing simultaneously acquired digital images of the two fluorophores. Here we demonstrate the simplicity and effectiveness of the FLAP method in revealing both fast and slow molecular dynamics in living cells using a Zeiss LSM 510 laser scanning confocal microscope.

Keywords:

  • Fluorescence microscopy;
  • laser scanning confocal microscopy;
  • FLAP;
  • GFP;
  • photobleaching;
  • actin;
  • cell motility