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Technical University of Munich

24 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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Medicine

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology
Lucia M.A. Crane 1, George Themelis 2, K. Tim Buddingh 1, Niels J. Harlaar 1, Rick G. Pleijhuis 1, Athanasios Sarantopoulos 2, Ate G.J. van der Zee 3, Vasilis Ntziachristos 2, Gooitzen M. van Dam 1
1Department of Surgery, Division of Surgical Oncology, University Medical Center Groningen, 2Helmholtz Zentrum, Technical University Munich, 3Department of Obstetrics and Gynaecology, University Medical Center Groningen

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

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Medicine

In vivo Near Infrared Fluorescence (NIRF) Intravascular Molecular Imaging of Inflammatory Plaque, a Multimodal Approach to Imaging of Atherosclerosis
Marcella A. Calfon 1, Amir Rosenthal 1,2, Georgios Mallas 1,3, Adam Mauskapf 1, R. Nika Nudelman 2, Vasilis Ntziachristos 2, Farouc A. Jaffer 1
1Cardiovascular Research Center and Cardiology Division, Massachusetts General Hospital, Harvard Medical School, 2Institute for Biological and Medical Imaging, Helmholtz Zentrum München und Technische Universität München, 3Department of Electrical and Computer Engineering, Northeastern University

We detail a new near-infrared fluorescence (NIRF) catheter for 2-dimensional intravascular molecular imaging of plaque biology in vivo. The NIRF catheter can visualize key biological processes such as inflammation by reporting on the presence of plaque-avid activatable and targeted NIR fluorochromes. The catheter utilizes clinical engineering and power requirements and is targeted for application in human coronary arteries. The following research study describes a multimodal imaging strategy that utilizes a novel in vivo intravascular NIRF catheter to image and quantify inflammatory plaque in proteolytically active inflamed rabbit atheromata.

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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

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology
Xiaofeng Cui *1,2,3, Guifang Gao *2,4, Tomo Yonezawa 5,6, Guohao Dai 1
1Department of Biomedical Engineering, Rensselaer Polytechnic Institute, 2Stemorgan Inc., 3Institute of Advanced Study, Technical University of Munich, 4Institute of Virology, School of Medicine, Wuhan University, 5Department of Molecular and Experimental Medicine, The Scripps Research Institute, 6Research Institute for Biomedical Sciences, Tokyo University of Science

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

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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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Medicine

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery
Rick Pleijhuis 1, Arwin Timmermans 1, Johannes De Jong 1, Esther De Boer 1, Vasilis Ntziachristos 2, Gooitzen Van Dam 1
1Department of Surgery, University Medical Center Groningen, 2Helmholtz Zentrum Munich, Technical University of Munich

Near-infrared fluorescence (NIRF) imaging may improve therapeutic outcome of breast cancer surgery by enabling intraoperative tumor localization and evaluation of surgical margin status. Using tissue-simulating breast phantoms containing fluorescent tumor-simulating inclusions, potential clinical applications of NIRF imaging in breast cancer patients can be assessed for standardization and training purposes.

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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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Biology

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies
Matthias Garten 1, Sophie Aimon 2, Patricia Bassereau 1, Gilman E. S. Toombes 3
1Institut Curie, Centre de Recherche, CNRS, UMR 168, PhysicoChimie Curie, Université Pierre et Marie Curie, 2Kavli Institute for Brain and Mind, University of California, San Diego, 3Molecular Physiology and Biophysics Section, National Institute for Neurological Disorders and Stroke, National Institute of Health

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods — electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

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Study on the Moisture Absorption and Desorption Rate of Bamboo Scrimber in a Hot-Humid Climatic Wind Tunnel
Zujian Huang 1,2,3, Yimin Sun 1,2, Florian Musso 3
1School of Architecture, South China University of Technology, 2State Key Laboratory of Subtropical Building Science, 3Chair of Building Construction and Material Science, Department of Architecture, Technical University of Munich

Here, we present a protocol to measure the moisture absorption and desorption rate of bamboo scrimber in an outdoor environment for a dynamic hot-humid climate wind tunnel test with complete meteorological conditions for 72 h.

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Biology

Subtype-specific Optical Action Potential Recordings in Human Induced Pluripotent Stem Cell-derived Ventricular Cardiomyocytes
Alexander Goedel *1,2, Dorota M. Zawada *1, Fangfang Zhang 1, Zhifen Chen 3, Alessandra Moretti 1,2, Daniel Sinnecker 1,2
1Medical Department I, University Hospital Klinikum rechts der Isar, Technical University of Munich, 2German Centre for Cardiovascular Research (DZHK), Munich Heart Alliance, 3Beth Israel Deaconess Medical Center, Harvard Medical School

Here we present a method to optically image action potentials, specifically in ventricular-like induced pluripotent stem cell-derived cardiomyocytes. The method is based on the promoter-driven expression of a voltage-sensitive fluorescent protein.

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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

3D Imaging of Soft-Tissue Samples using an X-ray Specific Staining Method and Nanoscopic Computed Tomography
Madleen Busse *1, Mark Müller *1, Melanie A. Kimm 2, Simone Ferstl 1, Sebastian Allner 1, Klaus Achterhold 1, Julia Herzen 1, Franz Pfeiffer 1,2
1Department of Physics and Munich School of BioEngineering, Technical University of Munich, 2Department of Diagnostic and Interventional Radiology, School of Medicine and Klinikum rechts der Isar, Technical University of Munich

A protocol for 3D visualization of microscopic tissue structures by using an X-ray specific staining method designed for X-ray computed tomography is presented.

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Neuroscience

Preparing Adult Drosophila melanogaster for Whole Brain Imaging during Behavior and Stimuli Responses
Alexandra Woller 1, Paul Bandow 1, Sophie Aimon 1, Ilona C. Grunwald Kadow 1
1Chair of Neuronal Control and Metabolism, School of Life Sciences, Technical University of Munich

We present a method specifically tailored to image the whole brain of adult Drosophila during behavior and in response to stimuli. The head is positioned to allow optical access to the whole brain, while the fly can move its legs and the antennae, the tip of the proboscis, and the eyes can receive sensory stimuli.

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Medicine

Development and Evaluation of 3D-Printed Cardiovascular Phantoms for Interventional Planning and Training
Maximilian Grab 1,2, Carina Hopfner 3, Alena Gesenhues 4, Fabian König 1,2, Nikolaus A. Haas 3, Christian Hagl 1, Adrian Curta 4, Nikolaus Thierfelder 1
1Department of Cardiac Surgery, Ludwig Maximilian University Munich, 2Chair of Medical Materials and Implants, Technical University of Munich, 3Department Pediatric Cardiology and Pediatric Intensive Care, Ludwig Maximilian University Munich, 4Department of Radiology, Ludwig Maximilian University Munich

Here we present development of a mock circulation setup for multimodal therapy evaluation, pre-interventional planning, and physician-training on cardiovascular anatomies. With the application of patient-specific tomographic scans, this setup is ideal for therapeutic approaches, training, and education in individualized medicine.

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Cancer Research

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells
Florian Stritzke 1,2, Hendrik Poeck 1,2,3,4, Simon Heidegger 1,2
1Department of Medicine III, School of Medicine, Technical University of Munich, 2Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, 3Department of Internal Medicine III, University Hospital Regensburg, 4National Centre for Tumor Diseases WERA

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

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Medicine

Combining 3D-Printing and Electrospinning to Manufacture Biomimetic Heart Valve Leaflets
Benedikt Freystetter 1, Maximilian Grab 1,2, Linda Grefen 1, Lara Bischof 1, Lorenz Isert 3, Petra Mela 2, Deon Bezuidenhout 4, Christian Hagl 1,5, Nikolaus Thierfelder 1
1Department of Cardiac Surgery, Ludwig Maximilians University Munich, 2Chair of Medical Materials and Implants, Technical University Munich, 3Faculty for Chemistry and Pharmacy, Ludwig Maximilians University Munich, 4Cardiovascular Research Unit, University of Cape Town, 5DZHK (German Centre for Cardiovascular Research), partner site Munich Heart Alliance

The presented method offers an innovative way for engineering biomimetic fiber structures in three-dimensional (3D) scaffolds (e.g., heart valve leaflets). 3D-printed, conductive geometries were used to determine shape and dimensions. Fiber orientation and characteristics were individually adjustable for each layer. Multiple samples could be manufactured in one setup.

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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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Neuroscience

Microfluidics-Assisted Selective Depolarization of Axonal Mitochondria
Simone Wanderoy *1,2, Alina Rühmkorf *1,2, Angelika B. Harbauer 2,3,4
1TUM Medical Graduate Center, Technical University of Munich, 2Max Planck Institute of Neurobiology, 3TUM School of Medicine, Institute of Neuronal Cell Biology, Technical University of Munich, 4Munich Cluster for Systems Neurology

The present protocol describes the seeding and staining of neuronal mitochondria in microfluidic chambers. The fluidic pressure gradient in these chambers allows for the selective treatment of mitochondria in axons to analyze their properties in response to pharmacological challenges without affecting the cell body compartment.

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Cancer Research

Syngeneic Mouse Orthotopic Allografts to Model Pancreatic Cancer
Constantin Schmitt 1,2,3, Dieter Saur 1,2,3,4, Stefanie Bärthel *1,2,3, Chiara Falcomatà *1,2,3
1Division of Translational Cancer Research, German Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), 2Translational Cancer Research and Institute of Experimental Cancer Therapy, Klinikum rechts der Isar, School of Medicine, Technical University of Munich, 3Center for Translational Cancer Research (TranslaTUM), School of Medicine, Technical University of Munich, 4Department of Internal Medicine II, Klinikum rechts der Isar, Technical University of Munich

Syngeneic mouse orthotopic allografts of pancreatic ductal adenocarcinoma (PDAC) recapitulate the biology, phenotypes, and therapeutic responses of disease subtypes. Owing to their fast, reproducible tumor progression, they are widely used in preclinical studies. Here, we show common practices to generate these models, injecting syngeneic murine PDAC cultures into the pancreas.

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Biology

Isolation of Nuclei from Flash-Frozen Liver Tissue for Single-Cell Multiomics
Mateusz Strzelecki 1,4, Kelvin Yin 1, Carlos Talavera-López 2,3, Celia P. Martinez-Jimenez 1,4
1Helmholtz Pioneer Campus (HPC), Helmholtz Munich, 2Institute of Computational Biology, Computational Health Department, Helmholtz Munich, 3Division of Infectious Diseases and Tropical Medicine, Ludwig-Maximillian-Universität Klinikum, 4TUM School of Medicine, Technical University of Munich

Here, we present a protocol for isolating nuclei from flash-frozen, archived liver tissues for single-nucleus RNA-seq, ATAC-seq, and joint multiomics (RNA-seq and ATAC-seq).

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Medicine

Performing Repeated Intraoperative Impedance Telemetry Measurements during Cochlear Implantation
Nora M. Weiss 1,2,3,4,5, Stefan Hans 6, Martin Wozniak 6, Aline Föger 6, Stefan Dazert 1, Vincent Van Rompaey 2,7, Paul Van de Heyning 2,7, Joachim Schmutzhard 8, Angelika Dierker 6
1Department of Otorhinolaryngology-Head and Neck Surgery, Ruhr-University Bochum, St. Elisabeth-Hospital Bochum, 2Department of Translational Neurosciences, Faculty of Medicine and Health Sciences, University of Antwerp, 3International Graduate School of Neuroscience (IGSN), Ruhr-University Bochum, 4Technical University of Munich, 5Department of Otorhinolaryngology, TUM School of Medicine, Klinikum rechts der Isar, 6MED-EL, 7Department of Otorhinolaryngology and Head & Neck Surgery, Antwerp University Hospital, 8Department of Otorhinolaryngology-Head and Neck Surgery, Medical University Innsbruck

Here we present a protocol to conduct repeated impedance telemetry measurements during cochlear implantation (CI). They may allow conclusions on the electrode's and implant's function. Repeated impedance measurements enable objective feedback on whether the electrode is positioned inside the perilymph or outside the inner ear.

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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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