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Analysis of heterogeneous fluorescence photobleaching by video kinetics imaging: The method of cumulants
Author(s) -
Koppel Dennis E.,
Carlson Curt,
Smilowitz Henry
Publication year - 1989
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.1989.tb02882.x
Subject(s) - photobleaching , fluorescence , fluorescein , autofluorescence , chemistry , fluorescence lifetime imaging microscopy , kinetics , biological system , optics , physics , biology , quantum mechanics
SUMMARY The method of cumulants has been applied to digital video fluorescence microscopy. The method is used to reconstruct the distribution of fluorescent molecules before the initiation of fluorescence photobleaching, and to characterize heterogeneous photobleaching by imaging one or more of the cumulants of the bleaching decay rate. Using the pipelined pixel processor of the image analysis system for the bulk of the calculations, rather than the general‐purpose host‐computer CPU, the video kinetics imaging can be performed in near real‐time. The method is applied to chick embryo myotubes labelled with fluorescein‐conjugated α‐bungarotoxin. The pre‐bleach fluorescence distribution is derived, and the image of fluorescein fluorescence is separated from glutaraldehyde‐induced autofluorescence on the basis of the spatially resolved average photobleaching decay rate.