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

TitleThe Number of Accumulated Photons and the Quality of Stimulated Emission Depletion Lifetime Images
Publication TypeJournal Article
Year of Publication2014
AuthorsSyed, A, Lesoine, MD, Bhattacharjee, U, Petrich, JW, Smith, EA
JournalPhotochemistry and Photobiology
Volume90
Pagination767-772
Date Published07
Type of ArticleArticle
ISBN Number0031-8655
Accession NumberWOS:000339096400005
Keywordsactin, binding, breaking, fluorescence microscopy, limit, phalloidin, resolution, sted microscopy
Abstract

Time binning is used to increase the number of photon counts in the peak channel of stimulated emission depletion 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 similar to 40 nm. This corresponds to a 10-fold 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 40-400 nm spatial regime.

DOI10.1111/php.12248
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