FLIM is a very versatile microscopy method where fluorescence lifetime information is combined with spatial localization in the sample, allowing investigating, for example, biochemical and physical processes, detecting changes in the local environment of the sample, molecular interactions, or conformational changes via Förster Resonance Energy Transfer (FRET). FLIM data acquisition is typically based 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 considered 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 in TCSPC modules with ultra short dead times and hybrid photomultiplier detector assemblies. More than 10 frames per second can be acquired, depending on sample brightness and image size. These improved hardware components enable higher detection count rates, making it possible to achieve much better photon statistics in shorter time spans.
With the new method, 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.