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University of Florence

14 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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Medicine

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples
Raffaele Coppini 1, Cecila Ferrantini 2, Alessandro Aiazzi 2, Luca Mazzoni 1, Laura Sartiani 1, Alessandro Mugelli 1, Corrado Poggesi 2, Elisabetta Cerbai 1
1Department NeuroFarBa, Division of Pharmacology, University of Florence, 2Department of Clinical and Experimental Medicine, Division of Physiology, University of Florence

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

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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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Neuroscience

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations
Valerio Conti *1, Aurelie Carabalona *2,3,15, Emilie Pallesi-Pocachard 2,3,4, Richard J. Leventer 5,6,7, Fabienne Schaller 2,3,8, Elena Parrini 1, Agathe A. Deparis 2,3, Françoise Watrin 2,3, Emmanuelle Buhler 2,3,8, Francesca Novara 9, Stefano Lise 10, Alistair T. Pagnamenta 10, Usha Kini 11, Jenny C. Taylor 10, Orsetta Zuffardi 9,12, Alfonso Represa 2,3, David Antony Keays 13, Renzo Guerrini 1,14, Antonio Falace 2,3, Carlos Cardoso 2,3
1University of Florence, 2INSERM INMED, 3Aix-Marseille University, 4Plateforme Biologie Moléculaire et Cellulaire INMED, 5Royal Children's Hospital, 6Murdoch Children's Research Institute, 7University of Melbourne, 8Plateforme postgenomique INMED, 9University of Pavia, 10Wellcome Trust Centre for Human Genetics, 11Oxford Radcliffe NHS Trust, 12IRCCS Casimiro Mondino Foundation, 13Research Institute of Molecular Pathology, 14IRCCS Stella Maris, 15Columbia University

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

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Engineering

Free-form Light Actuators — Fabrication and Control of Actuation in Microscopic Scale
Hao Zeng 1, Piotr Wasylczyk 2, Camilla Parmeggiani 1,3, Daniele Martella 1,4, Diederik Sybolt Wiersma 1
1European Labratory for Non-Linear Spectroscopy, University of Florence, 2Institute of Experimental Physics, Faculty of Physics, University of Warsaw, 3CNR-INO, 4Chemistry Department, University of Florence

Here, we fabricate 3D polymeric micro/nano structures in which both the shape and the molecular alignment can be engineered with nanometer scale accuracy by the use of direct laser writing. Light induced deformation of several types of liquid crystalline elastomer microstructures can be controlled in the microscopic scale.

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Cancer Research

Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma
Gaia Palmini 1, Roberto Zonefrati 1, Carmelo Mavilia 1, Alessandra Aldinucci 2, Ettore Luzi 1, Francesca Marini 1, Alessandro Franchi 1, Rodolfo Capanna 3, Annalisa Tanini 1, Maria Luisa Brandi 1
1Department of Surgery and Translational Medicine (DCMT), University of Florence, 2Neurofarba Department, University of Florence, 3Department of Traumatology and General Orthopedics, Azienda Ospedaliera Universitaria Careggi

The presence of cancer stem cells (CSCs) in bone sarcomas has recently been linked to their pathogenesis. In this article, we present the isolation of CSCs from primary cell cultures obtained from human biopsies of conventional osteosarcoma (OS) using the ability of CSCs to grow under nonadherent conditions.

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Bioengineering

An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging
Giulia Matrone 1, Alessandro Ramalli 2, Alessandro Stuart Savoia 3, Fabio Quaglia 4, Gloria Castellazzi 5, Patrizia Morbini 6, Marco Piastra 1
1Department of Electrical, Computer and Biomedical Engineering, University of Pavia, 2Department of Information Engineering, University of Florence, 3Department of Engineering, Roma Tre University, 4FTMTR&D/SPA, STMicroelectronics, 5Brain Connectivity Center, BCC, Istituto Neurologico Nazionale Fondazione C. Mondino I.R.C.C.S., 6Department of Molecular Medicine - Unit of Pathology, University of Pavia, Foundation IRCCS Policlinico San Matteo

The development of new ultrasound (US) probes based on Capacitive Micromachined Ultrasonic Transducer (CMUT) technology requires an early realistic assessment of imaging capabilities. We describe a repeatable experimental protocol for US image acquisition and comparison with magnetic resonance images, using an ex vivo bovine brain as an imaging target.

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Immunology and Infection

Investigating Aortic Valve Calcification via Isolation and Culture of T Lymphocytes using Feeder Cells from Irradiated Buffy Coat
Lavinia Curini *1,2, Mary Roxana Christopher *1, Herko Grubitzsch 3,4, Ulf Landmesser 1,3, Amedeo Amedei *2,5, Alexander Lauten *1,6, Brunilda Alushi *1,6
1Department of Cardiology, Campus Benjamin Franklin, Charité Universitätsmedizin Berlin and German Centre for Cardiovascular Research (DZHK), 2Department of Experimental and Clinical Medicine, University of Florence, 3Berlin Institute of Health, 4Department of Cardiology, German Heart Centre Berlin (DHZB), 5Sod of Interdisciplinary Internal Medicine, Azienda Ospedaliera Universitaria Careggi (AOUC), 6Department of General and Interventional Cardiology, Helios Klinikum Erfurt

In this study, we describe the process of T lymphocyte isolation from fresh samples of calcified aortic valves and the analytical steps of T cell-cloning for the characterization of the adaptive leukocyte subsets by using flow cytometry analysis.

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Chemistry

Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor
Letizia Barbieri 1,2, Enrico Luchinat 1,3
1Magnetic Resonance Center − CERM, University of Florence, 2Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine − CIRMMP, 3Neurofarba Department, University of Florence

This protocol describes the setup of an NMR bioreactor to keep encapsulated human cells viable for up to 72 h, followed by time-resolved in-cell NMR data acquisition and analysis. The methodology is applied to monitor intracellular protein-ligand interactions in real time.

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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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Cancer Research

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection
Tiziano Lottini 1, Matteo Buonamici 1, Claudia Duranti 1, Annarosa Arcangeli 1
1Department of Experimental and Clinical Medicine, Section of Internal Medicine, University of Florence

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

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iNEXT-Discovery and Instruct-ERIC: Integrating High-End Services for Translational Research in Structural Biology
Hans Wienk 1, Lucia Banci 2, Susan Daenke 3, Eva Pereiro 4, Harald Schwalbe 5, David Ian Stuart 6, Manfred S. Weiss 7, Anastassis Perrakis 1
1Division of Biochemistry and Oncode Institute, The Netherlands Cancer Institute, Amsterdam, 2CERM/CIRMMP, University of Florence, 3Instruct-ERIC, Oxford University, 4MISTRAL beamline, Alba Light Source, 5Center for Biomolecular Magnetic Resonance (BMRZ), Goethe University Frankfurt, 6Division of Structural Biology, Oxford University, 7Macromolecular Crystallographyy, Helmholtz-Zentrum Berlin

iNEXT-Discovery and Instruct-ERIC: Integrating High-End Services for Translational Research in Structural Biology

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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 Cheli 1, Luca Pesce 1,3, Niamh Brady 1, Giacomo Mazzamuto 1,4,5, Irene Costantini 1,4,6, Francesco S. Pavone 1,4,5
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.

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