UNIT 21.2 Fluorescence Localization After Photobleaching (FLAP)
Published Online: 1 OCT 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 OCT 2004
- Published Print: SEP 2004
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.
- Fluorescence microscopy;
- laser scanning confocal microscopy;
- cell motility