Fluorescence localization after photobleaching (FLAP).

Graham A. Dunn*, Mark R. Holt, Daniel Y.H. Soong, Colin Gray, Daniel Zicha

*Corresponding author for this work

Research output: Contribution to journalReview articlepeer-review

6 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)Unit 21.2
JournalCurrent protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
VolumeChapter 21
Publication statusPublished - Oct 2004

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