Interleaved gate acquisition: a low-frequency noise-immune approach to wide field FLIM with time-gated SPAD cameras

Fluorescence lifetime imaging microscopy (FLIM) data acquisition using time-gated single-photon avalanche diode (SPAD) cameras with megapixel sizes involves the sequential acquisition of data at a number G of different gate locations to sample the whole fluorescence decay. This requirement of multiple, high signal-to-noise ratio (SNR) gate images acquisition, which may take several hundred of ms or longer, limits the achievable FLIM frame-rate, but also make them susceptible to artefacts when the signal’s intensity is fluctuating faster than the gate series can be acquired. Here, we implement a data acquisition scheme designed to counteract this effect by rapidly acquiring complete sequences of low SNR gate images at the maximum rate sustainable by the camera (50 kHz per 1-bit gate image), allowing a full 1-bit resolution decay to be acquired in G x 20 μs. By repeating this sequence of gate shifts N times and accumulating the corresponding gate images, decays with arbitrary SNR can be obtained at a rate of 1/(N x G x 20 μs), that are immune to any signal intensity fluctuations happening at any frequency smaller than 1/(G x 20 μs) or 0.78 kHz. We illustrate the efficiency of this acquisition approach (dubbed interleaved gate acquisition) with SwissSPAD2 using simple examples.