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12 ARTICLES PUBLISHED IN JoVE

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Biology

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging
Claudio Vinegoni 1,2, Daniel Razansky 3, Jose-Luiz Figueiredo 1,2, Lyuba Fexon 1,2, Misha Pivovarov 1,2, Matthias Nahrendorf 1,2, Vasilis Ntziachristos 3, Ralph Weissleder 1,2
1Center for Systems Biology, Harvard Medical School, 2Center for Systems Biology, MGH - Massachusetts General Hospital, 3Institute for Biological and Medical Imaging, Technical University of Munich and Helmholtz Center Munich

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

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Biology

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila
Claudio Vinegoni 1, Daniel Razansky 2, Chrysoula Pitsouli 3, Norbert Perrimon 3, Vasilis Ntziachristos 2, Ralph Weissleder 1
1Center for Systems Biology, Massachusetts General Hospital, 2Institute for Biological and Medical Imaging (IBMI), Technical University of Munich and Helmholtz Center Munich, 3Department of Genetics, Harvard Medical School and Howard Hughes Medical Institute

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

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Bioengineering

Visualization of Cortex Organization and Dynamics in Microorganisms, using Total Internal Reflection Fluorescence Microscopy
Felix Spira 1, Julia Dominguez-Escobar 1, Nikola Müller 1,2, Roland Wedlich-Söldner 1
1AG Cellular Dynamics and Cell Patterning, Max Planck Institute of Biochemistry, 2Helmholtz Zentrum München

Total Internal Reflection Fluorescence (TIRF) microscopy is a powerful approach to observe structures close to the cell surface at high contrast and temporal resolution. We demonstrate how TIRF can be employed to study protein dynamics at the cortex of cell wall-enclosed bacterial and fungal cells.

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Immunology and Infection

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation
Sebastian C. Warth 1, Vigo Heissmeyer 1
1Institute for Molecular Immunology, Helmholtz Zentrum München

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

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Bioengineering

Wideband Optical Detector of Ultrasound for Medical Imaging Applications
Amir Rosenthal 1, Stephan Kellnberger 1, Murad Omar 1, Daniel Razansky 1, Vasilis Ntziachristos 1
1Institute for Biological and Medical Imaging (IBMI), Technical University of Munich and Helmholtz Center Munich

Optical detection of ultrasound is impractical in many imaging scenarios because it often requires stable environmental conditions. We demonstrate an optical technique for ultrasound sensing in volatile environments with miniaturization and sensitivity levels appropriate for optoacoustic imaging in restrictive scenarios, e.g. intravascular applications.

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Bioengineering

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time
Xosé Deán-Ben 1, Thomas Felix Fehm 1,2, Daniel Razansky 1,2
1Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, 2Faculty of Medicine, Technische Universität München

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

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Cancer Research

Hyperpolarized 13C Metabolic Magnetic Resonance Spectroscopy and Imaging
Eugen Kubala *1,2,3, Kim A. Muñoz-Álvarez *1, Geoffrey Topping 1, Christian Hundshammer 1,2, Benedikt Feuerecker 1, Pedro A. Gómez 3,4, Giorgio Pariani 1,5,6, Franz Schilling 1, Steffen J. Glaser 2, Rolf F. Schulte 3, Marion I. Menzel 3, Markus Schwaiger 1
1Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universität München, 2Department of Chemistry, Technische Universität München, 3GE Global Research, 4Zentralinstitut für Medizintechnik der Technischen Universität München (IMETUM), Technische Universität München, 5Institute for Biological and Medical Imaging (IBMI), Helmholtz Zentrum München, 6IDG Institute of Developmental Genetics, Helmholtz Zentrum München

Dynamic nuclear polarization with subsequent sample dissolution has enabled real-time studies of metabolism in biological systems. Hyperpolarized [1-13C]pyruvate was used to study lactate dehydrogenase activity in a prostate carcinoma cell line in vitro.

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Developmental Biology

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration
Jonathan M. Tsai 1,2, Irving L Weissman 1,2, Yuval Rinkevich 3,4
1Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 2Department of Developmental Biology, Stanford University School of Medicine, 3Comprehensive Pneumology Center, Institute of Lung Biology and Disease, Helmholtz Zentrum München, 4German Center for Lung Research (DZL)

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

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Developmental Biology

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon
Tamara Durovic 1,2, Jovica Ninkovic 1,3,4
1Institute of Stem Cell Research, Helmholtz Zentrum München, 2Graduate School of Systemic Neurosciences, Ludwig Maximilian University, 3Department of Cell Biology and Anatomy, Biomedical Center, Ludwig Maximilian University, 4Munich Cluster for Systems Neurology (SyNergy)

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

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Biology

Visualizing Scar Development Using SCAD Assay - An Ex-situ Skin Scarring Assay
Pushkar Ramesh 1, Haifeng Ye 1, Bikram Dasgupta 1, Hans-Günther Machens 2, Yuval Rinkevich 1
1Institute of Regenerative Biology and Medicine, Helmholtz Zentrum München, 2School of Medicine, Klinikum Rechts der Isar, Department of Plastic and Hand Surgery, Technical University of Munich

This protocol describes the generation of a skin-fascia explant termed "SCar like tissue in A Dish" or SCAD. This model allows unprecedented visualization of single fibroblasts during scar formation.

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Biochemistry

Single-Copy Gene Locus Chromatin Purification in Saccharomyces cerevisiae
Atiqa Sajid 1, Stephan Hamperl 1
1Institute of Epigenetics and Stem Cells, Helmholtz Zentrum München

This protocol presents a locus-specific chromatin isolation method based on site-specific recombination to purify a single-copy gene locus of interest in its native chromatin context from budding yeast, Saccharomyces cerevisiae.

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Genetics

Application of Unsupervised Multi-Omic Factor Analysis to Uncover Patterns of Variation and Molecular Processes Linked to Cardiovascular Disease
Corinna Losert 1,2, Kami Pekayvaz 3,4, Viktoria Knottenberg 3,4, Leo Nicolai 3,4, Konstantin Stark 3,4, Matthias Heinig 1,2,4
1Institute of Computational Biology, German Research Center for Environmental Health, Helmholtz Zentrum München, 2Department of Computer Science, TUM School of Computation, Information and Technology, Technical University of Munich, 3Medizinische Klinik und Poliklinik I University Hospital Ludwig-Maximilian University, 4German Centre for Cardiovascular Research (DZHK), partner site Munich Heart Alliance

We present a flexible, extendible Jupyter-lab-based workflow for the unsupervised analysis of complex multi-omics datasets that combines different pre-processing steps, estimation of the multi-omics factor analysis model, and several downstream analyses.

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