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Picosecond time‐resolved microspectrofluorometry in live cells exemplified by complex fluorescence dynamics of popular probes ethidium and cyan fluorescent protein
Author(s) -
Tramier M.,
Kemnitz K.,
Durieux C.,
CoppeyMoisan M.
Publication year - 2004
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.1365-2818.2004.01271.x
Subject(s) - fluorescence , picosecond , cyan , full width at half maximum , streak camera , optics , time resolved spectroscopy , temporal resolution , fluorescence microscope , chemistry , materials science , physics , laser
Summary Time‐resolved microspectrofluorometry in live cells, based on time‐ and space‐correlated single‐photon counting, is a novel method to acquire spectrally resolved fluorescence decays, simultaneously in 256 wavelength channels. The system is calibrated with a full width at half maximum (FWHM) of 90 ps for the temporal resolution, a signal‐to‐noise ratio of 10 6 , and a spectral resolution of 30 (Δλ/Λ). As an exemple, complex fluorescence dynamics of ethidium and cyan fluorescent protein (CFP) in live cells are presented. Free and DNA intercalated forms of ethidium are simultaneously distinguishable by their relative lifetime (1.7 ns and 21.6 ns) and intensity spectra (shift of 7 nm). By analysing the complicated spectrally resolved fluorescence decay of CFP, we propose a fluorescence kinetics model for its excitation/desexcitation process. Such detailed studies under the microscope and in live cells are very promising for fluorescence signal quantification.