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 s. 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 s), that are immune to any signal intensity fluctuations happening at any frequency smaller than 1/(G x 20 s) or 0.78 kHz. We illustrate the efficiency of this acquisition approach (dubbed interleaved gate acquisition) with SwissSPAD2 using simple examples. Please verify that (1) all pages are present, (2) all figures are correct, (3) all fonts and special characters are correct, and (4) all text and figures fit within the red margin lines shown on this review document.