Spectrally resolved and high speed TCSPC-based fluorescence lifetime imaging (Conference Presentation)

Abstract

Using Time-Correlated Single Photon Counting (TCSPC) for the purpose of fluorescence lifetime measurements is often limited in speed due to pile-up and dead-time artifacts. This is particularly critical in fast imaging applications. With modern instrumentation this limitation can be lifted by reducing the dead-time of the TCSPC electronics to the absolute minimum imposed by the speed of the electrical detector signals. Another, complementing approach to speedy image acquisition is parallelization by means of simultaneous, time tagged readout of many detector channels. This of course puts high demands on the data throughput of the TCSPC system. Here we present a new integrated design, providing up to 8 independent input channels, an extremely short dead-time, very high time tagging throughput over USB 3.0, and a timing resolution of 80 ps. Apart from design features and benchmark results of the instrument as such, we show application results from spectrally resolved (sFLIM) and high speed confocal fluorescence lifetime imaging (rapidFLIM). We put special focus on life science applications, paving the way to monitor sub-second dynamics in live cell imaging, including lifetime based Forster Resonant Energy Transfer (FLIM-FRET) imaging. We furthermore show how the inevitable pulse-pile-up occurring in the detector signals at high photon flux can be corrected for and how this data acquisition scheme excels in terms of photon collection efficiency in comparison to other approaches.