Two-photon image-scanning microscopy with SPAD array and blind image reconstruction

Sami V Koho (1), Eli Slenders (1), Giorgio Tortarolo, Marco Castello, Mauro Buttafava, Federica Villa, Elena Tcarenkova, Marcel Ameloot, Paolo Bianchini, Colin JR Sheppard, Alberto Diaspro, Alberto Tosi, Giuseppe Vicidomini (see publication in Journal )


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 single-photon avalanche diode (SPAD) array detector and a novel blind image reconstruction method, is shown to enhance the effective resolution, as well as the overall image quality of 2PE microscopy. With our adaptive pixel reassignment procedure ~1.6 times resolution increase is maintained deep into thick semi-transparent samples. The integration of Fourier ring correlation based semi-blind deconvolution is shown to further enhance the effective resolution by a factor of sqrt(2) – and automatic background correction is shown to boost the image...