Compact and effective photon-resolved image scanning microscope

Fluorescence confocal laser-scanning microscopy (LSM) is one of the most popular tools for life science research. This popularity is expected to grow thanks to single-photon array detectors tailored for LSM. These detectors offer unique single-photon spatiotemporal information, opening new perspectives for gentle and quantitative superresolution imaging. However, a flawless recording of this information poses significant challenges for the microscope data acquisition (DAQ) system. We present a DAQ module based on the digital frequency domain principle, able to record essential spatial and temporal features of photons. We use this module to extend the capabilities of established imaging techniques based on single-photon avalanche diode (SPAD) array detectors, such as fluorescence lifetime image scanning microscopy. Furthermore, we use the module to introduce a robust multispecies approach encoding the fluorophore excitation spectra in the time domain. Finally, we combine time-resolved stimulated emission depletion microscopy with image scanning microscopy, boosting spatial resolution. Our results demonstrate how a conventional fluorescence laser scanning microscope can transform into a simple, information-rich, superresolved imaging system with the simple addition of a SPAD array detector with a tailored data acquisition system. We expected a blooming of advanced single-photon imaging techniques, which effectively harness all the sample information encoded in each photon.