Over the last two decades, time-resolved fluorescence microscopy has become an essential tool in Life Sciences thanks to measurement procedures such as Fluorescence Lifetime Imaging (FLIM), lifetime based Foerster Resonance Energy Transfer (FRET), and Fluorescence (Lifetime) Correlation Spectroscopy (F(L)CS) down to the single molecule level. Today, complete turn-key systems are available either as stand-alone units or as upgrades for confocal laser scanning microscopes (CLSM). Data acquisition on such systems is typically based on Time-Correlated Single Photon Counting (TCSPC) electronics along with picosecond pulsed diode lasers as excitation sources and highly sensitive, single photon counting detectors.
Up to now, TCSPC data acquisition is considered a somewhat slow process as a large number of photons per pixel is required for reliable data analysis, making it difficult to use FLIM for following fast FRET processes, such as signal transduction pathways in cells or fast moving sub-cellular structures. We present here a novel and elegant solution to tackle this challenge.
Our approach, named rapidFLIM, exploits recent hardware developments such as TCSPC modules with ultra short dead times and hybrid photomultiplier detector assemblies enabling significantly higher detection count rates. Thanks to these improved components, it is possible to achieve much better photon statistics in significantly shorter time spans while being able to perform FLIM imaging for fast processes in a qualitative manner and with high optical resolution. FLIM imaging can now be performed with up to several frames per second making it possible to study fast processes such as protein interactions involved in endosome trafficking.
Sandra Orthaus-Mueller, Benedikt Kraemer, Astrid Tannert, Tino Roehlicke, Michael Wahl, Hans-Juergen Rahn, Felix Koberling, and Rainer Erdmann, "Rapid FLIM: The new and innovative method for ultra-fast imaging of biological processes (Conference Presentation)," Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 1006919 (Presented at SPIE BiOS: January 31, 2017; Published: 24 April 2017); https://doi.org/10.1117/12.2249757.5380018905001.
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