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A compact real-time fluorescence lifetime imaging microscopy (FLIM) system based on an array of low dark count 0.13microm CMOS single-photon avalanche diodes (SPADs) is demonstrated. Fast background-insensitive fluorescence lifetime determination is achieved by use of a recently proposed algorithm called 'Integration for Extraction Method' (IEM) [J. Opt. Soc. Am. A 25, 1190 (2008)]. Here, IEM is modified for a wider resolvability range and implemented on the FPGA of the new SPAD array imager. We experimentally demonstrate that the dynamic range and accuracy of calculated lifetimes of this new camera is suitable for widefield FLIM applications by imaging a variety of test samples, including various standard fluorophores covering a lifetime range from 1.6ns to 16ns, microfluidic mixing of fluorophore solutions, and living fungal spores of Neurospora Crassa. The calculated lifetimes are in a good agreement with literature values. Real-time fluorescence lifetime imaging is also achieved, by performing parallel 32 x 16 lifetime calculations, realizing a compact and low-cost FLIM camera and promising for bigger detector arrays.

Real-time fluorescence lifetime imaging system with a 32 × 32 013μm CMOS low dark-count single-photon avalanche diode array