Time-Resolved STED Microscopy with Single-Photon Detector Array: a Perfect Synergy

Image Scanning Microscopy (ISM) [1] successfully overcomes the trade-off between resolution and signal-to-noise ratio of traditional confocal microscopes by considering the spatial distribution of the fluorescence emission light and by reassigning the detected photons accordingly (i.e., pixel-reassignment). A recent implementation [2] upgrades a confocal to an image scanning microscope by substituting the traditional single-element detector with a SPAD array detector. Notably, the SPAD array samples the fluorescence signal from the detection/probing volume both in space (such as cameras) and in time (such as single element detectors, e.g., SPADs), providing potentially significant extra information for a variety of experimental contexts. To fully exploit this advantage, we present a versatile FPGA-based time-resolved microscopy platform that parallelly acquires all the SPAD array signals with a sub-nanosecond temporal resolution, thanks to a Digital Frequency Domain architecture. In the context of stimulated emission depletion (STED) microscopy, we leverage the platform to decrease the STED power needed to achieve a target spatial resolution [3] . In particular, we show how to synergically exploit both the spatial and temporal extra information to implement a new and dedicated photon-reassignment method for STED microscopy. This method not only takes advantage of the pixel-reassignment principle but also compensates for all the different sources of background which typically reduce resolution and imaging quality in STED microscopy, i.e., incomplete depletion [4] , [5] , direct-excitation from the STED beam and out-of-focus signal. Additionally, the platform allows for fluorescence lifetime imaging and a straightforward pulsed interleaved excitation (PIE) implementation, enabling dual-color STED microscopy ( Fig. 1 ).