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National Institute of Optics, National Research Council

5 ARTICLES PUBLISHED IN JoVE

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Neuroscience

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy
Ludovico Silvestri 1, Alessandro Bria 2,3, Irene Costantini 1, Leonardo Sacconi 1,4, Hanchuan Peng 5, Giulio Iannello 2, Francesco Saverio Pavone 1,4,6,7
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.

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Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo
Anna L. Allegra Mascaro 1, Leonardo Sacconi 1,2, Francesco Saverio Pavone 1,2,3,4
1European Laboratory for Non-Linear Spectroscopy, University of Florence, 2National Institute of Optics, National Research Council, 3Department of Physics and Astronomy, University of Florence, 4International Center for Computational Neurophotonics (ICON Foundation)

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

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Biology

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions
Carina Monico 1,2, Gionata Belcastro 1, Francesco Vanzi 1,3, Francesco S. Pavone 1,4,5,6, Marco Capitanio 1,4
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.

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Biology

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy
L. Gardini 1,2, A. V. Kashchuk 2,3, F. S. Pavone 1,2,3, M. Capitanio 2,3
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.

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Biology

Mesoscopic Optical Imaging of Whole Mouse Heart
Francesco Giardini *1, Erica Lazzeri *1, Camilla Olianti *1, Giada Beconi 1, Irene Costantini 1,2, Ludovico Silvestri 1,3,4, Elisabetta Cerbai 1,5, Francesco S. Pavone 1,3,4, Leonardo Sacconi 1,3,6
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.

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