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Robust approaches to quantitative ratiometric FRET imaging of CFP/YFP fluorophores under confocal microscopy.
Abstract
Ratiometric quantification of CFP/YFP FRET enables live-cell time-series detection
of molecular interactions, without the need for acceptor photobleaching or specialized
equipment for determining fluorescence lifetime. Although popular in widefield applications,
its implementation on a confocal microscope, which would enable sub-cellular resolution,
has met with limited success. Here, we characterize sources of optical variability
(unique to the confocal context) that diminish the accuracy and reproducibility of
ratiometric FRET determination and devise practical remedies. Remarkably, we find
that the most popular configuration, which pairs an oil objective with a small pinhole
aperture, results in intractable variability that could not be adequately corrected
through any calibration procedure. By quantitatively comparing several imaging configurations
and calibration procedures, we find that significant improvements can be achieved
by combining a water objective and increased pinhole aperture with a uniform-dye calibration
procedure. The combination of these methods permitted remarkably consistent quantification
of sub-cellular FRET in live cells. Notably, this methodology can be readily implemented
on a standard confocal instrument, and the dye calibration procedure yields a time
savings over traditional live-cell calibration methods. In all, identification of
key technical challenges and practical compensating solutions promise robust sub-cellular
ratiometric FRET imaging under confocal microscopy.
Type
Journal articleSubject
Cell LineFluorescence Resonance Energy Transfer
Fluorescent Dyes
Humans
Image Processing, Computer-Assisted
Microscopy, Confocal
Staining and Labeling
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https://hdl.handle.net/10161/15557Published Version (Please cite this version)
10.1111/j.1365-2818.2008.03109.xPublication Info
Tadross, MR; Park, SA; Veeramani, B; & Yue, DT (2009). Robust approaches to quantitative ratiometric FRET imaging of CFP/YFP fluorophores
under confocal microscopy. J Microsc, 233(1). pp. 192-204. 10.1111/j.1365-2818.2008.03109.x. Retrieved from https://hdl.handle.net/10161/15557.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Michael Raphael Tadross
Assistant Professor of Biomedical Engineering
Dr. Tadross' lab develops technologies to rapidly deliver drugs to genetically defined
subsets of cells in the brain. By using these reagents in mouse models of neuropsychiatric
disease, his group is mapping how specific receptors on defined cells and synapses
in the brain give rise to diverse neural computations and behaviors. The approach
leverages drugs currently in use to treat human neuropsychiatric disease, facilitating
clinically relevant interpretation of the mapping effort.<

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