Exploiting the detector distance information in image scanning microscopy by phasor-based SPLIT-ISM

Confocal microscopy is an important bio-imaging technique that increases the resolution using a spatial pinhole to block out-of-focus light. In theory, the maximum resolution and optical sectioning are obtained when the detection pinhole is fully closed, but this is prevented by the dramatic decrease in the signal reaching the detector. In image scanning microscopy (ISM) this limitation is overcome by the use of an array of point detectors rather than a single detector. This, combined with pixel reassignment, increases the resolution of √2 over widefield imaging, with relatively little modification to the existing hardware of a laser-scanning microscope. Separation of photons by lifetime tuning (SPLIT) is a super-resolution technique, based on the phasor analysis of the fluorescent signal into an additional channel of the microscope. Here, we use SPLIT to analyze the information encoded within the array detectors distance for improving the resolution of ISM (SPLIT-ISM). We find that the lateral resolution can be increased of an additional 1.3 × with respect to the pixel-reassigned image with a concomitant increase in optical sectioning. We applied the SPLIT-ISM technique on biological images acquired by two currently available ISM systems: the Genoa Instruments PRISM and the Zeiss Airyscan. We evaluate the improvement provided by SPLIT-ISM through the QuICS algorithm, a quantitative tool based on image correlation spectroscopy. QuICS allows extracting three parameters related to the resolution, and contrast SNR of the image. We find that SPLIT-ISM provides an increase in spatial resolution for both the Genoa Instrument PRISM and the Zeiss Airyscan microscopes.