Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain

Year: 2012

Authors: Silvestri L., Bria A., Sacconi L., Iannello G., Pavone FS.

Autors Affiliation: European Laboratory for Non-Linear Spectroscopy (LENS), University of Florence, Italy; Integrated Research Centre, University Campus Bio-Medico of Rome, Italy; Department of Automation, Electromagnetism, Information Engineering and Industrial Mathematics (DAEMI), University of Cassino, Italy; National Institute of Optics, National Research Council, Italy; Department of Physics, University of Florence, Italy; International Center of Computational Neurophotonics (ICON), Italy

Abstract: Elucidating the neural pathways that underlie brain function is one of the greatest challenges in neuroscience. Light sheet based microscopy is a cutting edge method to map cerebral circuitry through optical sectioning of cleared mouse brains. However, the image contrast provided by this method is not sufficient to resolve and reconstruct the entire neuronal network. Here we combined the advantages of light sheet illumination and confocal slit detection to increase the image contrast in real time, with a frame rate of 10 Hz. In fact, in confocal light sheet microscopy (CLSM), the out-of-focus and scattered light is filtered out before detection, without multiple acquisitions or any post-processing of the acquired data. The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies. (C) 2012 Optical Society of America

Journal/Review: OPTICS EXPRESS

Volume: 20 (18)      Pages from: 20582  to: 20598

More Information: We thank Dr. Anna Letizia Allegra Mascaro and Dr. Francesco Vanzi for helpful discussion about the manuscript, Dr. Filippo Biamonte, Dr. Filippo Giorgi and Dr. Claudia Laperchia for assistance in perfusions, Mr. Riccardo Ballerini for mechanical assistance, Prof. Flavio Keller for providing us with L7-GFP mice. The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) un der grant agreement n. 228334. This research project has been also supported by Human Frontier Science Program research grant (RGP0027/2009), and by the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX. This research has been carried out in the framework of the research activities of ICON foundation supported by Ente Cassa di Risparmio di Firenze.
KeyWords: Plane Illumination Microscopy; Optical Sectioning Microscopy; Structured-illumination; Embryonic-development; Infantile-autism; Transgenic Mice; Purkinje-cells; Reconstruction; Contrast; Ultramicroscopy
DOI: 10.1364/OE.20.020582

Citations: 188
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