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National Institute of Optics, National Research Council

5 ARTICLES PUBLISHED IN JoVE

Neuroscience

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Ludovico Silvestri¹, Alessandro Bria^2,3, Irene Costantini¹, Leonardo Sacconi^1,4, Hanchuan Peng⁵, Giulio Iannello², 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.

Neuroscience

Laser Nanosurgery of Cerebellar Axons In Vivo

Anna L. Allegra Mascaro¹, 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.

Biology

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

Carina Monico^1,2, Gionata Belcastro¹, 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.

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.

Biology

Mesoscopic Optical Imaging of Whole Mouse Heart

Francesco Giardini^*1, Erica Lazzeri^*1, Camilla Olianti^*1, Giada Beconi¹, 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.