Global analysis of fluorescence lifetime imaging microscopy data.

Abstract:

:Global analysis techniques are described for frequency domain fluorescence lifetime imaging microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Verveer PJ,Squire A,Bastiaens PI

doi

10.1016/S0006-3495(00)76759-2

subject

Has Abstract

pub_date

2000-04-01 00:00:00

pages

2127-37

issue

4

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(00)76759-2

journal_volume

78

pub_type

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