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 m...

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Veröffentlicht in:Biomedical optics express 2020-06, Vol.11 (6), p.2905-2924
Hauptverfasser: Koho, Sami V., Slenders, Eli, Tortarolo, Giorgio, Castello, Marco, Buttafava, Mauro, Villa, Federica, Tcarenkova, Elena, Ameloot, Marcel, Bianchini, Paolo, Sheppard, Colin J. R., Diaspro, Alberto, Tosi, Alberto, Vicidomini, Giuseppe
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container_end_page 2924
container_issue 6
container_start_page 2905
container_title Biomedical optics express
container_volume 11
creator Koho, Sami V.
Slenders, Eli
Tortarolo, Giorgio
Castello, Marco
Buttafava, Mauro
Villa, Federica
Tcarenkova, Elena
Ameloot, Marcel
Bianchini, Paolo
Sheppard, Colin J. R.
Diaspro, Alberto
Tosi, Alberto
Vicidomini, Giuseppe
description 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.
doi_str_mv 10.1364/BOE.374398
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title Two-photon image-scanning microscopy with SPAD array and blind image reconstruction
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