Journal
JOURNAL OF BIOMEDICAL OPTICS
Volume 6, Issue 3, Pages 287-291Publisher
SPIE-SOC PHOTO-OPTICAL INSTRUMENTATION ENGINEERS
DOI: 10.1117/1.1383063
Keywords
fluorescence resonance energy transfer; protein interactions; wide-field; confocal; two-photon; lifetime imaging; FRET microscopy; CAATT/enhancer binding protein alpha (C/EBP alpha)
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Fluorescence resonance energy transfer (FRET) microscopy is a better method than the x-ray diffraction, nuclear magnetic resonance, or electron microscopy for studying the structure and localization of proteins under physiological conditions. In this paper, we describe four different light microscopy techniques to visualize the interactions of the transcription factor CAATT/enhancer binding protein alpha (C/EBP alpha) in living pituitary cells. In wide-field, confocal, and two-photon microscopy the FRET image provides two-dimensional spatial distribution of steady-state protein-protein interactions. The two-photon imaging technique provides a better FRET signal (less bleedthrough and photobleaching) compared to the other two techniques. This information, although valuable, falls short of revealing transient interactions of proteins in real time. The fluorescence lifetime methods allow us to monitor FRET signals at the moment of the protein interactions at a resolution on the order of subnanoseconds, providing high temporal, as well as spatial resolution. This paper will provide a brief review of the above-mentioned FRET techniques. (C) 2001 Society of Photo-Optical Instrumentation Engineers.
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