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

Authors:

Koho, S. V., Slenders, E., Tortarolo, G., Castello, M., Buttafava, M., Villa, F., ... & Vicidomini, G.

Keywords:

Detector arrays, Fluorescence lifetime imaging, Image quality, Multiphoton microscopy, Scanning microscopy, Structured illumination microscopy

Abstract:

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 ∼2 – and automatic background correction is shown to boost the image quality especially in noisy images. Most importantly, our 2PE-ISM implementation requires no calibration measurements or other input from the user, which is an important aspect in terms of day-to-day usability of the technique.

< Back to publications

Full article >