Journal
IEEE TRANSACTIONS ON IMAGE PROCESSING
Volume 14, Issue 9, Pages 1237-1245Publisher
IEEE-INST ELECTRICAL ELECTRONICS ENGINEERS INC
DOI: 10.1109/TIP.2005.852458
Keywords
confocal microscopy; energy migration fluorescence resonance energy transfer (emFRET); enhanced green fluorescent protein (eGFP); scale selective filtering
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Anisotropy imaging can be used to image resonance energy transfer between pairs of identical fluorophores and, thus, constitutes a powerful tool for monitoring protein homo-association in living single cells. The requirement for only a single fluorophore significantly simplifies biological preparation and interpretation. We use quantitative methods for the acquisition and image processing of anisotropy data that return the expected error of the anisotropy per pixel based on photon statistics. The analysis methods include calibration procedures and allow for a balance in spatial, anisotropy, and temporal resolution. They are featured here with anisotropy images of fluorescent calibration beads and enhanced green fluorescent protein complexes in live cells.
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