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Issue 1, 2005
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Time-resolved fluorescence microscopy

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In fluorescence microscopy, the fluorescence emission can be characterised not only by intensity and position, but also by lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM) can report on photophysical events that are difficult or impossible to observe by fluorescence intensity imaging, and time-resolved fluorescence anisotropy imaging (TR-FAIM) can measure the rotational mobility of a fluorophore in its environment. We compare different FLIM methods: a chief advantage of wide-field time-gating and phase modulation methods is the speed of acquisition whereas for time-correlated single photon counting (TCSPC) based confocal scanning it is accuracy in the fluorescence decay. FLIM has been used to image interactions between proteins such as receptor oligomerisation and to reveal protein phosphorylation by detecting fluorescence resonance energy transfer (FRET). In addition, FLIM can also probe the local environment of fluorophores, reporting, for example, on the local pH, refractive index, ion or oxygen concentration without the need for ratiometric measurements.

Graphical abstract: Time-resolved fluorescence microscopy

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Article information

20 Aug 2004
04 Oct 2004
First published
11 Nov 2004

Photochem. Photobiol. Sci., 2005,4, 13-22
Article type

Time-resolved fluorescence microscopy

K. Suhling, P. M. W. French and D. Phillips, Photochem. Photobiol. Sci., 2005, 4, 13
DOI: 10.1039/B412924P

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