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Technische Universität München

25 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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Neuroscience

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction
Monika S. Brill 1, Petar Marinković 1, Thomas Misgeld 1,2,3,4
1Lehrstuhl für Biomolekulare Sensoren, Technische Universität München, 2Center for Integrated Protein Science (Munich) at the Institute of Neuroscience, Technische Universität München, 3TUM Institute for Advanced Study and German Center for Neurodegenerative Diseases, Technische Universität München, 4Munich Cluster for Systems Neurology (SyNergy), Technische Universität München

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

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Biology

Naïve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures
Vera Wenzel 1, Daniela Roedl 1, Johannes Ring 2, Karima Djabali 1
1Department of Dermatology and Institute for Medical Engineering, Technische Universität München, 2Department of Dermatology and Allergology, Technische Universität München

We report a method to isolate naïve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

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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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Medicine

Simulating Pancreatic Neuroplasticity: In Vitro Dual-neuron Plasticity Assay
Ihsan Ekin Demir 1, Elke Tieftrunk 1, Karl-Herbert Schäfer 2, Helmut Friess 1, Güralp O. Ceyhan 1
1Department of Surgery, Klinikum rechts der Isar, Technische Universität München, 2Department of Biotechnology, University of Applied Sciences Kaiserslautern/Zweibrücken

Neuronal plasticity is an increasingly recognized, but insufficiently understood feature of the gastrointestinal (GI) tract. Here, in the example of human pancreatic disorders, we present an in vitro neuroplasticity assay for the study of neuronal plasticity in the GI tract at both morphological and functional level.

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Bioengineering

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo
Thilo L. Schenck 1, Myra N. Chávez 1, Alexandru P. Condurache 2, Ursula Hopfner 1, Farid Rezaeian 3, Hans-Günther Machens 1, José T. Egaña 1,4
1Department of Plastic Surgery and Hand Surgery, University Hospital rechts der Isar, Technische Universität München, 2Institute for Signal Processing, University of Lübeck, 3Department of Plastic Surgery and Hand Surgery, University Hospital Zürich, 4FONDAP Center for Genome Regulation, Facultad de Ciencias, Universidad de Chile

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

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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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Medicine

Ischemic Tissue Injury in the Dorsal Skinfold Chamber of the Mouse: A Skin Flap Model to Investigate Acute Persistent Ischemia
Yves Harder 1, Daniel Schmauss 1, Reto Wettstein 2, José T. Egaña 1, Fabian Weiss 1, Andrea Weinzierl 1, Anna Schuldt 1, Hans-Günther Machens 1, Michael D. Menger 3, Farid Rezaeian 4
1Department of Plastic Surgery and Hand Surgery, Klinikum rechts der Isar, Technische Universität München, 2Department of Plastic, Reconstructive, Aesthetic and Hand Surgery, University Hospital of Basel, 3Institute for Clinical and Experimental Surgery, University of Saarland, 4Division of Plastic and Hand Surgery, University Hospital Zurich

The window of the murine dorsal skinfold chamber presented visualizes a zone of acute persistent ischemia of a musculocutaneous flap. Intravital epi-fluorescence microscopy permits for direct and repetitive assessment of the microvasculature and quantification of hemodynamics. Morphologic and hemodynamic results can further be correlated with histological and molecular analyses.

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Engineering

Investigating Single Molecule Adhesion by Atomic Force Spectroscopy
Frank W. S. Stetter 1,2, Sandra Kienle 1,2, Stefanie Krysiak 1,2, Thorsten Hugel 1,2
1Physik-Department E22a, Technische Universität München, 2IMETUM, Technische Universität München

A protocol to couple a large variety of single molecules covalently onto an AFM tip is presented. Procedures and examples to determine the adhesion force and free energy of these molecules on solid supports and bio-interfaces are provided.

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Medicine

Heterotopic Cervical Heart Transplantation in Mice
Thomas Ratschiller 1,4, Marcus-André Deutsch 1, Julia Calzada-Wack 2, Frauke Neff 2, Christiane Roesch 3, Ralf Guenzinger 3, Ruediger Lange 3, Markus Krane 3
1Department of Cardiovascular Surgery, Division of Experimental Surgery, German Heart Center Munich, Technische Universität München, 2Institute of Pathology, Helmholtz Zentrum Munich, 3Department of Cardiovascular Surgery, Division of Experimental Surgery, German Heart Center Munich, Technische Universität München, 4Department of Cardiovascular and Thoracic Surgery, General Hospital Linz

The cuff technique shortens and facilitates the model of heterotopic cervical heart transplantation in mice by avoiding technically challenging suture anastomoses of small vessels. The technical details shown in this video paper should allow researches to establish this model in their laboratories.

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JoVE Journal

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis
Broder Rühmann 1, Jochen Schmid 1, Volker Sieber 1
1Chair of Chemistry of Biogenic Resources, Technische Universität München

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

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Biology

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles
Harald Lahm *1, Stefanie A. Doppler *1, Martina Dreßen 1, Klaudia Adamczyk 1, Marcus-André Deutsch 1, Hanna Ulrich 2, Matthias Schiemann 2,3, Rüdiger Lange 1,4, Markus Krane 1,4
1Department of Cardiovascular Surgery, German Heart Center Munich, Technische Universität München, 2Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München, 3Clinical Cooperation Groups: "Antigen-specific Immunotherapy" and "Immune Monitoring", Helmholtz Center Munich (Neuhererg), Technische Universität München, 4DZHK (German Center for Cardiovascular Research) – Partner site Munich Heart Alliance

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

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Developmental Biology

Imaging Subcellular Structures in the Living Zebrafish Embryo
Peter Engerer 1, Gabriela Plucinska 1,2, Rachel Thong 1,3, Laura Trovò 1, Dominik Paquet 4,5,6, Leanne Godinho 1
1Institute of Neuronal Cell Biology, Technische Universität München, 2Cell Biology, Department of Biology, Faculty of Science, Utrecht University, 3Faculty of Biology, Ludwig-Maximilians-Universität-München, 4Adolf-Butenandt-Institute, Biochemistry, Ludwig-Maximilians-Universität-München, 5German Center for Neurodegenerative Diseases, 6Laboratory of Brain Development and Repair, The Rockefeller University

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

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Medicine

Transarterial Administration of Oncolytic Viruses for Locoregional Therapy of Orthotopic HCC in Rats
Jennifer Altomonte 1, Kim A. Muñoz-Álvarez 2, Katsunori Shinozaki 3, Christine Baumgartner 4, Georgios Kaissis 5, Rickmer Braren 5, Oliver Ebert 1
1II. Medizinische Klinik, Klinikum rechts der Isar der TU München, 2Department of Nuclear Medicine, Klinikum rechts der Isar der TU München, 3Division of Clinical Oncology, Hiroshima Prefectural Hospital, 4Center for Preclinical Research, Klinikum rechts der Isar der TU München, 5Institute for Diagnostic and Interventional Radiology, Klinikum rechts der Isar der TU München

Oncolytic virotherapies are under development as novel therapeutics for the treatment of hepatocellular carcinoma (HCC). Here we describe a method for locoregional therapy of HCC via hepatic arterial administration of oncolytic virus.

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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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Biochemistry

Modification and Functionalization of the Guanidine Group by Tailor-made Precursors
Tobias G. Kapp *1, Maximilian Fottner *1, Horst Kessler 1
1Department of Chemistry, Technische Universität München

A protocol for the synthesis of alkyl-modified guanidines based on the use of the corresponding precursors is presented.

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Genetics

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection
Eric K. Hubner 1,2, Christian Lechler 1, Thomas N. Rösner 1, Birgit Kohnke-Ertel 1, Roland M. Schmid 1, Ursula Ehmer 1
1Department of Medicine II, Klinikum rechts der Isar, Technische Universität München, 2Department of Pneumology, Center for Medicine, Medical Center University of Freiburg

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

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Biochemistry

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions
Markus Götz 1, Philipp Wortmann 1, Sonja Schmid 1,2, Thorsten Hugel 1
1Institute of Physical Chemistry, University of Freiburg, 2Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

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Biochemistry

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments
Louisa Jutta Dietz 1, Anuroop Venkateswaran Venkatasubramani 1, Annika Müller-Eigner 2, Martin Hrabe de Angelis 3,4,5, Axel Imhof 1, Lore Becker 3, Shahaf Peleg 2,6,7
1Munich Center of Integrated Protein Science and Biomedical Center, Ludwig-Maximilians University of Munich, 2Laboratory for Metabolism and Epigenetics in Aging, Leibniz Institute for Farm Animal Biology (FBN), 3German Mouse Clinic, Helmholtz Zentrum Munich, German Research Center for Environment and Health (GmbH), 4German Center for Diabetes Research (DZD), 5Chair of Experimental Genetics, School of Life Science Weihenstephan, Technische Universität München, 6Laboratory for Metabolism and Epigenetics in Brain Aging, Institute of Neuroregeneration & Neurorehabilitation of Qingdao University, 7Molecular Biology Division, Biomedical Center, Faculty of Medicine, Ludwig-Maximilians University of Munich

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

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Bioengineering

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography
Günther Pardatscher *1, Matthaeus Schwarz-Schilling *1, Sandra Sagredo 1, Friedrich C. Simmel 1
1Physics Department, Technical University of Munich

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

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Bioengineering

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth
Kilian Vogele 1, Thomas Frank 1, Lukas Gasser 1, Marisa A. Goetzfried 1, Mathias W. Hackl 2, Stephan A. Sieber 2, Friedrich C. Simmel 1,3, Tobias Pirzer 1
1Physics of Synthetic Systems - E14, Physics-Department and ZNN, Technische Universität München, 2Departmant of Chemistry, Center for Integrated Protein Science (CIPSM), Technische Universität München, 3Nanosystems Initiative Munich

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

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Chemistry

Covalent Attachment of Single Molecules for AFM-based Force Spectroscopy
Adrianna Kolberg 1, Christiane Wenzel 1, Thorsten Hugel 1,3, Markus Gallei 2, Bizan N. Balzer 1,3
1Institute of Physical Chemistry, Albert-Ludwigs-Universität Freiburg, 2Chair in Polymer Chemistry, Saarland University, 3Cluster of Excellence livMatS at FIT - Freiburg Center for Interactive Materials and Bioinspired Technologies, University of Freiburg

Covalent attachment of probe molecules to atomic force microscopy (AFM) cantilever tips is an essential technique for the investigation of their physical properties. This allows us to determine the stretching force, desorption force and length of polymers via AFM-based single molecule force spectroscopy with high reproducibility.

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Immunology and Infection

Systematic Scoring Analysis for Intestinal Inflammation in a Murine Dextran Sodium Sulfate-Induced Colitis Model
Vicky Garcia-Hernandez 1, Philipp-Alexander Neumann 2, Stefan Koch 3,4, Renae Lyons 1, Asma Nusrat 1, Charles A. Parkos 1
1Department of Pathology, University of Michigan, 2Department of Surgery, Klinikum Rechts der Isar, Technische Universität München, 3Wallenberg Centre for Molecular Medicine, Linköping University, 4Department of Biomedical and Clinical Sciences (BKV), Linköping University

Systematic scoring of intestinal inflammation using a free computer-assisted system is a powerful tool to quantitatively compare histopathological changes in colitis models characterized by the presence of ulcers and inflammatory changes. Histological colitis score evaluation strengthens clinical observations and facilitates data interpretation.

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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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