Biosensor Forster resonance energy transfer detection by the phasor approach to fluorescence lifetime imaging microscopy.

ABSTRACT: We present here the phasor approach to biosensor Förster resonance energy transfer (FRET) detection by fluorescence lifetime imaging microscopy (FLIM) and show that this method of data representation is robust towards biosensor design as well as the fluorescence artifacts inherent to the cellular environment. We demonstrate this property on a series of dual and single chain biosensors, which report the localization of Rac1 and RhoA activity, whilst performing concomitant ratiometric FRET analysis on the acquired FLIM data by the generalized polarization (GP) approach. We then evaluate and compare the ability of these two methods to quantitatively image biosensor FRET signal as a function of time and space. We find that with lifetime analysis in the phasor plot each molecular species is tra

SUBMITTER: Hinde E 

PROVIDER: S-EPMC3523109 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature