Phasor imaging with a widefield photon-counting detector.

Journal of Biomedical Optics
Ryan A ColyerX Michalet

Abstract

Fluorescence lifetime can be used as a contrast mechanism to distinguish fluorophores for localization or tracking, for studying molecular interactions, binding, assembly, and aggregation, or for observing conformational changes via Förster resonance energy transfer (FRET) between donor and acceptor molecules. Fluorescence lifetime imaging microscopy (FLIM) is thus a powerful technique but its widespread use has been hampered by demanding hardware and software requirements. FLIM data is often analyzed in terms of multicomponent fluorescence lifetime decays, which requires large signals for a good signal-to-noise ratio. This confines the approach to very low frame rates and limits the number of frames which can be acquired before bleaching the sample. Recently, a computationally efficient and intuitive graphical representation, the phasor approach, has been proposed as an alternative method for FLIM data analysis at the ensemble and single-molecule level. In this article, we illustrate the advantages of combining phasor analysis with a widefield time-resolved single photon-counting detector (the H33D detector) for FLIM applications. In particular we show that phasor analysis allows real-time subsecond identification of species b...Continue Reading

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Citations

Dec 26, 2012·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·X MichaletS Cova
Mar 13, 2015·Biophysical Journal·WeiYue ChenClemens F Kaminski
Jan 23, 2020·Methods and Applications in Fluorescence·Arin UlkuXavier Michalet
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Mar 4, 2017·Biophysical Journal·Kyoungwon Park, Shimon Weiss

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