Confocal multispot microscope for fast and deep imaging in semicleared tissues

Year: 2018

Authors: Adam M.P., Muellenbroich M.C., Di Giovanna A.P., Alfieri D., Silvestri L., Sacconi L., Pavone F.S.

Autors Affiliation: University of Florence, European Laboratory for Non-linear Spectroscopy, Florence, Italy; National Institute of Optics, National Research Council, Florence, Italy; L4T-LIGHT4TECH Srl, Sesto Fiorentino, Italy; University of Florence, Department of Physics, Florence, Italy

Abstract: Although perfectly transparent specimens are imaged faster with light-sheet microscopy, less transpar- ent 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 demon- strate microscope performance in fixed brain slices by im- aging dendritic spines up to 400-μm deep.


Volume: 23 (2)      Pages from: 020503-1  to: 020503-4

More Information: This project received funding from the Italian Institute of Technology in Genoa and the European Union’s H2020 Research and Innovation Programme under Grant Agreements No. 720270 (Human Brain Project) and 654148 (Laserlab-Europe), and from the EU programme H2020 EXCELLENT SCIENCE, European Research Council (ERC) under Grant Agreement ID No. 692943 (BrainBIT). The project has also been supported by the Italian Ministry for Education, University, and Research in the framework of the Flagship Project NanoMAX and of Eurobioimaging Italian Nodes (ESFRI Research Infrastructure), and by Ente Cassa di Risparmio di Firenze (Private Foundation).
KeyWords: Microscopes; Optical transfer function; Photons, Acquisition time; Deep tissue imaging; Experimental evaluation; Light-sheet microscopies; Line detection; Microscope performance; Two photon microscopy; Two-photon excitations, Tissue
DOI: 10.1117/1.JBO.23.2.020503

Citations: 1
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