Beyond photobleaching, laser illumination unbinds fluorescent proteins.

TitleBeyond photobleaching, laser illumination unbinds fluorescent proteins.
Publication TypeJournal Article
Year of Publication2009
AuthorsHeinze KG, Costantino S, De Koninck P, Wiseman PW
JournalJ Phys Chem B
Volume113
Issue15
Pagination5225-33
Date Published2009 Apr 16
ISSN1520-6106
KeywordsAntibodies, Antigen-Antibody Reactions, Green Fluorescent Proteins, Lasers, Photobleaching, Photons
Abstract

Confocal and two-photon fluorescence microscopy techniques using genetically encoded fluorescent probes are widely used in cell biology. Beyond the common problems of photobleaching and phototoxicity, we present evidence that photounbinding also has the potential to compromise such methods, especially in quantitative studies. We show that laser intensities within excitation regimes typical for imaging approaches such as as fluorescence recovery after photobleaching (FRAP), photolysis, or fluorescence correlation spectroscopy (FCS) experiments can cause the dissociation of antibodies from their ligands. Indeed, both one- and two-photon excitation of a fluorescent anti-GFP antibody caused its dissociation from immobilized GFP in vitro. Importantly, with two-photon excitation, the laser intensity threshold for photobleaching was the same as for photounbinding. By contrast, with single-photon excitation, we found a range of laser intensities where photobleaching can be separated from photounbinding. This photounbinding effect was visualized and measured by rebinding a second fluorescent anti-GFP (Green Fluorescent Protein) antibody, indicating that the GFP remained functional for reassociation following the photoinduced dissociation. Finally, we show that this unbinding effect occurs only when at least one binding partner carries a fluorescent label. Our results show that this photounbinding effect can readily remain masked or be misinterpreted as photobleaching, which can compromise the quantitative interpretation of binding studies made using fluorescence microscopy.

DOI10.1021/jp8060152
Alternate JournalJ Phys Chem B
PubMed ID19309095

Funding

Our research endeavors are made possible by the following agencies:

Canadian Institutes of Health Research - Instituts de recherche en santé du Canada Fonds de recherche du Québec – Nature et technologies (FRQNT)Fonds de la recherche en santé du Québec NARSAD Natural Sciences and Engineering Research Council of Canada (NSERC) - Conseil de recherche en sciences naturelles et en génie du Canada (CRSNG)innovation.caHuman Frontier Science Program