Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 116, Issue 3, Pages 864-873Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1805333116
Keywords
photoswitchable; FRET; microscopy; fluorescence; imaging
Categories
Funding
- Intramural Research Program of the NIH
- National Institute of Biomedical Imaging and Bioengineering
- NATIONAL INSTITUTE OF BIOMEDICAL IMAGING AND BIOENGINEERING [ZIAEB000071] Funding Source: NIH RePORTER
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FRET is a powerful approach to study the interactions of fluorescent molecules, and numerous methods have been developed to measure FRET in cells. Here, we present a method based on a donor molecule's photoswitching properties, which are slower in the presence vs. the absence of an acceptor. The technique, photoswitching FRET (psFRET), is similar to an established but underutilized method called photobleaching FRET (pbFRET), with the major difference being that the molecules are switched off rather than photobleached. The psFRET technique has some of the FRET imaging advantages normally attributed to fluorescence lifetime imaging microscopy (FLIM), such as monitoring only donor fluorescence. However, it can be performed on a conventional widefield microscope, requires less illumination light to photoswitch off than photobleaching, and can be photoswitched on again to repeat the experiment. We present data testing the validity of the psFRET approach to quantify FRET in cells and demonstrate its use in imaging protein-protein interactions and fluorescent protein-based biosensors.
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