National Institute of Optics National Research Council 5 articles published in JoVE Neuroscience Optical Clearing and Labeling for Light-sheet Fluorescence Microscopy in Large-scale Human Brain Imaging Danila Di Meo*1, Josephine Ramazzotti*1, Marina Scardigli*1,2, Franco Cheli1, Luca Pesce1,3, Niamh Brady1, Giacomo Mazzamuto1,4,5, Irene Costantini1,4,6, Francesco S. Pavone1,4,5 1European Laboratory for Non-linear Spectroscopy (LENS), University of Florence, 2Division of Physiology, Department of Experimental and Clinical Medicine, University of Florence, 3Department of Physics, University of Pisa, 4National Research Council - National Institute of Optics (CNR-INO), 5Department of Physics and Astronomy, University of Florence, 6Department of Biology, University of Florence The present protocol provides a step-by-step procedure for rapid and simultaneous optical clearing, muti-round labeling, and 3D volumetric reconstruction of tens of postmortem human brain sections by combining the (SWITCH - H2O2 - Antigen Retrieval - 2,2'-thiodiethanol [TDE]) SHORT tissue transformation technique with light-sheet fluorescence microscopy imaging in a routinely high-throughput protocol. Biology Mesoscopic Optical Imaging of Whole Mouse Heart Francesco Giardini*1, Erica Lazzeri*1, Camilla Olianti*1, Giada Beconi1, Irene Costantini1,2, Ludovico Silvestri1,3,4, Elisabetta Cerbai1,5, Francesco S. Pavone1,3,4, Leonardo Sacconi1,3,6 1European Laboratory for Non-Linear Spectroscopy, 2Department of Biology, University of Florence, 3National Institute of Optics, National Research Council, 4Department of Physics and Astronomy, University of Florence, 5Department of Neurosciences, Psychology, Drugs and Child Health, University of Florence, 6Institute for Experimental Cardiovascular Medicine, Faculty of Medicine, University of Freiburg We report a method for mesoscopic reconstruction of the whole mouse heart by combining new advancements in tissue transformation and staining with the development of an axially scanned light-sheet microscope. Biology Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy L. Gardini1,2, A. V. Kashchuk2,3, F. S. Pavone1,2,3, M. Capitanio2,3 1National Institute of Optics, National Research Council, 2LENS, European Laboratory for Non-Linear Spectroscopy, 3Physics Department, University of Florence Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis. Biology Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions Carina Monico1,2, Gionata Belcastro1, Francesco Vanzi1,3, Francesco S. Pavone1,4,5,6, Marco Capitanio1,4 1LENS - European Laboratory for Non-linear Spectroscopy, University of Florence, 2Chemistry Research Laboratory, University of Oxford, 3Department of Biology, University of Florence, 4Department of Physics and Astronomy, University of Florence, 5National Institute of Optics-National Research Council, Italy, 6International Center of Computational Neurophotonics Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres. Neuroscience Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy Ludovico Silvestri1, Alessandro Bria2,3, Irene Costantini1, Leonardo Sacconi1,4, Hanchuan Peng5, Giulio Iannello2, Francesco Saverio Pavone1,4,6,7 1European Laboratory for Non-linear Spectroscopy (LENS), 2Integrated Research Centre, University Campus Bio-medico of Rome, 3DAEMI, University of Cassino, 4National Institute of Optics (CNR-INO), 5Allen Institute for Brain Science, 6Department of Physics, University of Florence, 7ICON Foundation, Sesto Fiorentino, Italy In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.
