Laser Scanning Microscopy with SPAD Array Detector: Towards a New Class of Fluorescence Microscopy Techniques

Laser scanning microscopy is one of the most common architectures in fluorescence microscopy. In a nutshell: the objective lens focuses the laser beam(s) and generates an effective excitation spot which is scanned on the sample; for each pixel, the fluorescent image is projected into a single-element detector, which – typically – spatially and temporally integrates the fluorescent light along its sensitive area and the pixel dwell-time, thus providing a single-intensity value per pixel. Notably, the integration performed by the single-element detector hinders any additional information potentially encoded in the dynamic and image of the fluorescent spot. To address this limitation, we recently upgraded the detection unit of a laser scanning microscope, replacing the single-element detector with a novel SPAD (single photon avalanche diode) array detector. We have shown at first that the additional spatial information allows to overcome the trade-off between resolution and signal-to-noise ratio proper of confocal microscopy: indeed, this architecture represents the natural implementation of image scanning microscopy (ISM). We then exploited the single-photon-timing ability of the SPAD array detector elements to combine ISM with fluorescence lifetime imaging: the results show higher resolution and better lifetime accuracy with respect to the confocal counterpart. Moreover, we explored the combination of our ISM platform with stimulated emission depletion (STED) microscopy, to mitigate the non-negligible chance of photo-damaging a sample. Lastly, we showed how the SPAD array-based microscope can be used in the context of single-molecule/particle tracking (SMT or SPT) and spectroscopy. Indeed, we implemented a real-time, feedback based SMT architecture which can potentially correlate the dynamics of a bio-molecule with its structural changes and micro-environment, taking advantage of the time-resolved spectroscopy ability of the novel detector. We believe that this novel laser scanning microscopy architecture has everything in its favour to substitute current single-element detector approaches; it will enable for a new class of fluorescence microscopy techniques capable of investigating complex living biological samples with unprecedented spatial and temporal characteristics and augmented information content.