We present an achromatic confocal laser scanning system capable of recording spectrally resolved fluorescence lifetime images (sFLIMs) at a rate of >8 frames per second (FPS) for a 128 x 128 image. This frame rate was achieved by optimizing the processing of lifetime calculations from previous results which demonstrated >4 FPS sFLIM imaging. The imaging system is achromatic for a spectral range of 400 - 900 nm, achieved by using reflective optics instead of a transmissive lens system, except for the primary objectives. Two excitation sources have been integrated into the system, 485 nm and 640 nm laser diodes with a pulse width of <70 ps and <90 ps respectively. Imaging is performed via a galvanometric mirror system which scans the laser beam over the sample with the ability to change the Field of View (FOV) on the fly. The collected fluorescence signal is focused into a multimode fiber via a second objective and recollimated onto a transmissive grating for spectral dispersion onto a novel complementary metal–oxide–semiconductor single photon avalanche diode (CMOS SPAD) line array sensor. This sensor can perform lifetime histogram generation on-chip and process over 16.5 Giga events/s enabling fast lifetime data acquisition. High speed sFLIM is demonstrated through imaging of convallaria majalis sections.
We demonstrate a 512 x 16 CMOS single photon avalanche diode (SPAD) line sensor with per-pixel on-chip histogramming for video rate spectral fluorescence lifetime imaging (sFLIM). On-chip histogramming provides 32-bin histograms per pixel with 11bit/bin dynamic range. In addition, bin widths in time can be programmed from 51.20 ps to 6.55 ns, providing a histogram range from 1.64 ns to 209.72 ns to suit a wide range of fluorescence decays. At the end of a user defined exposure time, the full histogram data (i.e. 32-bins/pixel and 512 pixels) is first transferred to a FPGA in 84.48 μs via 64 data I/O pads at a 33.33 MHz I/O rate. The sensor data is then binned into two user defined spectral bands to provide spectral separation between different fluorophores, before being transferred to a PC via a USB3 connection for further processing. Fluorescence lifetimes for each spectral band are then rapidly estimated in software by applying the Centre-of-Mass Method (CMM), providing two 128 x 128 size spectral lifetime images in 1.384 s (i.e. with a frame rate of 0.72 fps). The frame rate can be increased by reducing the number of bins, reaching a maximum frame rate when only 2 bins are used with the Rapid Lifetime Determination (RLD) algorithm. In this paper we study the lifetime accuracy vs frame rate trade-offs by varying the number of histogram bins while carefully adjusting the bin widths for maximum bin counts. We validate the results using a Rhodamine 110 and Rhodamine B mixture solution which we separate them spectrally by their fluorescence lifetimes.