University of Florence 14 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. Cancer Research Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection Tiziano Lottini1, Matteo Buonamici1, Claudia Duranti1, Annarosa Arcangeli1 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. 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. Chemistry Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor Letizia Barbieri1,2, Enrico Luchinat1,3 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. 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 Grubitzsch3,4, Ulf Landmesser1,3, 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. 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-Pocachard2,3,4, Richard J. Leventer5,6,7, Fabienne Schaller2,3,8, Elena Parrini1, Agathe A. Deparis2,3, Françoise Watrin2,3, Emmanuelle Buhler2,3,8, Francesca Novara9, Stefano Lise10, Alistair T. Pagnamenta10, Usha Kini11, Jenny C. Taylor10, Orsetta Zuffardi9,12, Alfonso Represa2,3, David Antony Keays13, Renzo Guerrini1,14, Antonio Falace2,3, Carlos Cardoso2,3 1University of Florence, 2INSERM INMED, 3Aix-Marseille University, 4Plateforme Biologie Moléculaire et Cellulaire INMED, 5 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. Bioengineering An Experimental Protocol for Assessing the Performance of New Ultrasound Probes Based on CMUT Technology in Application to Brain Imaging Giulia Matrone1, Alessandro Ramalli2, Alessandro Stuart Savoia3, Fabio Quaglia4, Gloria Castellazzi5, Patrizia Morbini6, Marco Piastra1 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. Cancer Research Establishment of Cancer Stem Cell Cultures from Human Conventional Osteosarcoma Gaia Palmini1, Roberto Zonefrati1, Carmelo Mavilia1, Alessandra Aldinucci2, Ettore Luzi1, Francesca Marini1, Alessandro Franchi1, Rodolfo Capanna3, Annalisa Tanini1, Maria Luisa Brandi1 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. Engineering Free-form Light Actuators — Fabrication and Control of Actuation in Microscopic Scale Hao Zeng1, Piotr Wasylczyk2, Camilla Parmeggiani1,3, Daniele Martella1,4, Diederik Sybolt Wiersma1 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. 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 Laser Nanosurgery of Cerebellar Axons In Vivo Anna L. Allegra Mascaro1, Leonardo Sacconi1,2, Francesco Saverio Pavone1,2,3,4 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. Medicine Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples Raffaele Coppini1, Cecila Ferrantini2, Alessandro Aiazzi2, Luca Mazzoni1, Laura Sartiani1, Alessandro Mugelli1, Corrado Poggesi2, Elisabetta Cerbai1 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. 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.