Fig. 1: Concepts of FLISM and FLFS.

a In FLISM, a pulsed laser beam is focused and scanned across the sample. For each position of the laser beam, the fluorescence is collected and focused onto a SPAD array detector. Every photon produces a pulse in one of the detection channels almost instantaneously. The BrightEyes-TTM measures the arrival time of the photon with respect to the last laser pulse and the pixel, line, and frame clock of the microscope. In single-point spot-variation FLFS, the laser beam is kept steady while the photon arrival times are measured. The movement of the fluorophores results in temporal fluctuations in the intensity trace. b A super-resolution fluorescence lifetime (FLISM) image can be reconstructed from the resulting 4D dataset (x, y, t, ch). For each time bin of the TCSPC histogram, a super-resolved ISM image is reconstructed with the adaptive pixel reassignment algorithm. All the images are then recombined, and the fluorescence lifetimes are calculated for each pixel, resulting in the final FLISM image. c In spot-variation FLFS, the diffusion time as a function of the focal spot area is measured. The dimension of the focal spot can be changed by combining the photon traces coming from different detection channels. From the autocorrelations of the resulting intensity time traces, the diffusion times, and hence the diffusion modality (free diffusion, diffusion through a meshwork, or diffusion in a sample comprising isolated microdomains), can be found. Simultaneously, from the start–stop times, the fluorescence lifetime τ_fl is measured. PCR photon count rate.