Confocal multispot microscope for fast and deep imaging in semicleared tissues

Marie-Pierre Adam; Marie Caroline Müllenbroich; Antonino Paolo Di Giovanna; Domenico Alfieri; Ludovico Silvestri; Leonardo Sacconi; Francesco Saverio Pavone

doi:10.1117/1.JBO.23.2.020503

19 February 2018 Confocal multispot microscope for fast and deep imaging in semicleared tissues

Marie-Pierre Adam, Marie Caroline Müllenbroich, Antonino Paolo Di Giovanna, Domenico Alfieri, Ludovico Silvestri, Leonardo Sacconi, Francesco Saverio Pavone

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Journal of Biomedical Optics, Vol. 23, Issue 2, 020503 (February 2018). https://doi.org/10.1117/1.JBO.23.2.020503

Abstract

Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transparent samples are often imaged with two-photon microscopy leveraging its robustness to scattering; however, at the price of increased acquisition times. Clearing methods that are capable of rendering strongly scattering samples such as brain tissue perfectly transparent specimens are often complex, costly, and time intensive, even though for many applications a slightly lower level of tissue transparency is sufficient and easily achieved with simpler and faster methods. Here, we present a microscope type that has been geared toward the imaging of semicleared tissue by combining multispot two-photon excitation with rolling shutter wide-field detection to image deep and fast inside semicleared mouse brain. We present a theoretical and experimental evaluation of the point spread function and contrast as a function of shutter size. Finally, we demonstrate microscope performance in fixed brain slices by imaging dendritic spines up to 400-μm deep.

CC BY: © The Authors. Published by SPIE under a Creative Commons Attribution 4.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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Marie-Pierre Adam, Marie Caroline Müllenbroich, Antonino Paolo Di Giovanna, Domenico Alfieri, Ludovico Silvestri, Leonardo Sacconi, and Francesco Saverio Pavone "Confocal multispot microscope for fast and deep imaging in semicleared tissues," Journal of Biomedical Optics 23(2), 020503 (19 February 2018). https://doi.org/10.1117/1.JBO.23.2.020503

Received: 20 October 2017; Accepted: 15 January 2018; Published: 19 February 2018

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