EASY TWO-PHOTON IMAGE-SCANNING MICROSCOPY WITH SPAD ARRAY AND BLIND IMAGE RECONSTRUCTION

Two-photon excitation (2PE) laser scanning microscopy is the imaging modality of choice when one desires to work with thick biological samples. However, its spatial resolution is poor, below confocal laser scanning microscopy. Here, we propose a straightforward implementation of 2PE image scanning microscopy (2PE-ISM) that, by leveraging our recently introduced ISM platform – based on a new single-photon avalanche diode (SPAD) array detector – coupled with a novel blind image reconstruction method, is shown to improve the effective resolution, as well as the overall image quality of 2PE microscopy. Indeed, in stark contrast to conventional single-point detectors, SPAD array detectors give access to the images of any excited scanning region, from which it is possible to decode information about the aberrations/distortions – occurring during imaging – able to substantially improve the reconstruction. Most importantly, our 2PE-ISM implementation requires no calibration or other input from the user; it works like any familiar two-photon system, but produces higher resolution images deep into thick samples. In our view, this novel implementation is the key for making 2PE-ISM mainstream.