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Title: The Number of Accumulated Photons and the Quality of Stimulated Emission Depletion Lifetime Images

Time binning is used to increase the number of photon counts in the peak channel of stimulated emission depletion (STED) fluorescence lifetime decay curves to determine how it affects the resulting lifetime image. The fluorescence lifetime of the fluorophore, Alexa Fluor 594 phalloidin, bound to F-actin is probed in cultured S2 cells at a spatial resolution of ~40 nm. This corresponds to a tenfold smaller probe volume compared to confocal imaging, and a reduced number of photons contributing to the signal. Pixel-by-pixel fluorescence lifetime measurements and error analysis show that an average of 40 ± 30 photon counts in the peak channel with a signal-to-noise ratio of 20 is enough to calculate a reliable fluorescence lifetime from a single exponential fluorescence decay. No heterogeneity in the actin cytoskeleton in different regions of the cultured cells was measured in the 40- to 400-nm spatial regime.
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  1. Ames Laboratory
Publication Date:
OSTI Identifier:
Report Number(s):
IS-J 8392
Journal ID: ISSN 0031-8655
DOE Contract Number:
Resource Type:
Journal Article
Resource Relation:
Journal Name: Photochemistry and Photobiology
The American Society of Photobiology
Research Org:
Ames Laboratory (AMES), Ames, IA (United States)
Sponsoring Org:
USDOE Office of Science (SC)
Country of Publication:
United States