Fluorescence Lifetime Imaging (FLIM) is a very versatile microscopy method wher
e fluorescence lifetime information is combined with spatial localization in the sample, allowing investigating, for example, biochemical and physical processes, detecting changes in the local enviro
nment of the sample, molecular interactions, or co
nformational changes via Förster Reso
nance Energy Transfer (FRET). FLIM data acquisition is typically ba
sed on Time-Correlated Single Photon Counting (TCSPC) electronics, pulsed diode lasers, and highly sensitive single photon counting detectors. Up to now TCSPC data acquisition is co
nsidered a somewhat slow process, due to the time required to collect a sufficient number of photons per pixel for reliable data analysis.
The novel rapidFLIM method allows acquiring several FLIM images per second by exploiting recent hardware developments such as TCSPC modules with ultra short dead times and hybrid photomultiplier detector assemblies. These improved hardware components enable higher detection count rates, making it possible to achieve much better photon statistics in shorter time spans. Thus, FLIM imaging can be performed for dynamic processes (e.g., transient protein interaction in living cells, chemical reaction, or ion flux) as well as highly mobile species in a precise manner and with high optical resolution.
As an additional benefit, the high accuracy of the data analysis is comparable to that of conventional TCSPC measurements.
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