Abstract
Image scanning microscopy (ISM) is emerging as the natural evolution of confocal laser-scanning microscopy, combining optical sectioning and a two-fold improvement in lateral resolution with high signal-to-noise ratio. Single-photon avalanche diode (SPAD) array detectors enable an effective implementation of ISM by recording a micro-image at each scan position with single-photon sensitivity, negligible read-out noise, and without the fixed frame-readout constraints of conventional cameras. Nevertheless, SPAD array-based ISM remains largely confined to laboratories with expertise in microscope development. Here, we present BrightEyes-Scope, a reference implementation of a SPAD array-based ISM system. We provide step-by-step guidelines for optical design, system construction, alignment, calibration, data acquisition, and image reconstruction. Presented as a complete ISM microscope, BrightEyes-Scope also serves as a reference for upgrading existing laser-scanning microscopes. We anticipate that it will accelerate the adoption of ISM for quantitative super-resolution imaging.