Live Biological Imaging Across Scales

STEFAN LINDER is Professor of Cellular Microbiology at University Medical Center Hamburg-Eppendorf (UKE), Germany. He is a member of the Life Science Committee at the Royal Microscopical Society, and he serves on the editorial boards of European Journal of Cell Biology (EJCB) and Frontiers in Immunology. CLAIRE M. WELLS is Professor of Cancer Cell Biology at Kings College London, UK. She is a member of the British Society for Cell Biology and a member of the Life Science Committee at the Royal Microscopical Society. She is Editor in Chief of Cell Adhesion and Migration.

• List of Contributors xiPreface xv1 Super- Resolution Imaging 1Alistair Curd, Amir Rahmani, Oliver Umney, Aleks Ponjavic, and Michelle Peckham1.1 Resolution, Widefield, and Confocal Imaging 11.2 SR- SIM: “Super- Resolution” Structured Illumination Using Spatially Varying Illumination 21.3 STED: Stimulated Emission Depletion Microscopy 51.4 Single Molecule Localisation Microscopies 71.5 Single- Molecule Light- Sheet Microscopy 111.6 Computation for Live- Cell Super Resolution 121.7 Advanced Applications in Cells 131.8 Concluding Remarks 14Acknowledgments 14References 142 Peering into the Septin Cytoskeleton – New Insights Unlocked by Microscopy Advances 23Andrew W. Schaefer and Elias T. Spiliotis2.1 Introduction – the Septin Cytoskeleton 232.2 Total Internal Reflection Fluorescence (TIRF) Microscopy Assays Elucidate Functions of Microtubule- and Actin- Associated Septins 252.3 Super- Resolution Fluorescence Microscopy Is Resolving the Nanoscale Organization of Membrane- and Actomyosin- Associated Septins 282.4 High- Speed Atomic Force Microscopy (HS- AFM) Reveals How Septins Assemble on Membranes 302.5 Cryo- Electron Tomography (Cryo- ET) Begins to Unveil the Supramolecular Organization of Septin Structures 322.6 Scoping the Future – Challenges and Needs of Septin Imaging 35Acknowledgments 36References 363 Imaging Phagocytosis: From Cells to Molecules 43Trieu Le, Ava Kavianpour, Spencer Freeman, and Sergio Grinstein3.1 Introduction 433.2 Fluorescence Labeling to Study Phagocytosis 453.2.1 Antibodies to Endogenous or Epitope- Tagged Proteins 453.2.2 Fluorescent Chimeras of Proteins 453.3 Pros and Cons of Microscopy Techniques to Examine Phagocytosis 463.3.1 Epifluorescence and Confocal Microscopy 463.3.2 Lattice Light- Sheet Microscopy 463.3.3 Frustrated Phagocytosis 473.4 Single- Particle Tracking 483.4.1 The Principle 483.4.2 Requirements of SPT 493.4.3 Motion Types 503.4.4 Physicochemical and Optical Properties of Fluorescent Probes Affecting SPT 513.4.4.1 Specificity 513.4.4.2 Valency 523.4.4.3 Size and Molecular Weight 523.4.4.4 Brightness 523.4.4.5 Blinking 523.4.4.6 Photostability 523.4.5 Photophysical Properties of Probes Commonly Used in SPT 553.4.5.1 Fluorescent Proteins 553.4.5.2 Organic Dyes 553.4.5.3 Quantum Dots 563.4.5.4 Protein Tags 563.4.6 Application of Fluorescent Probes in Labeling Biomolecules for SPT 573.4.7 Application of SPT to Study Phagocytosis 583.5 Concluding Remarks 58Acknowledgments 59References 604 Imaging Wound Healing Across Scales 65Simran Rawal and Tamal Das4.1 Introduction: Starting from Scratch 654.2 Visualizing Wound Healing in Real Time 674.3 Dynamics of Cell Migration During Wound Healing 694.3.1 Immune Cell Migration 694.3.2 Collective Migration of Epithelial Cells 724.4 ECM Remodeling 744.5 Conclusions and Perspectives 75Acknowledgments 76References 765 Leukocyte and Cancer Adhesion Live- Cell Imaging 81Camilla Cerutti5.1 Introduction 815.2 Live- Cell Imaging of Leukocyte Adhesion 825.3 Live- Cell Imaging of Cancer Adhesion 875.4 Concluding Remarks 91References 916 Bridging the Gap Between In Vitro and In Vivo Studies with Live- Cell Imaging of Organoids 97Francesco Pampaloni6.1 Introduction 976.1.1 Importance of Live Imaging to Analyze the Physiological Behavior of Cells and Tissues 976.1.2 Light Dose and Peak Intensity Determine Phototoxicity in the Specimen 986.1.3 Irradiance and Light Dose in 3D (Volumetric) Fluorescence Microscopy 1006.1.3.1 Laser Scanning Confocal Microscopy 1006.1.3.2 Spinning Disk Confocal Microscopy 1016.1.3.3 Multiphoton Microscopy 1016.1.3.4 Light- Sheet Fluorescence Microscopy 1036.1.4 Live Imaging with WFM 1056.1.5 Artificial Intelligence for Live Imaging 1056.2 Organoids: A Paradigm Change Bridging the Gap Between In Vivo and In Vitro Assays 1066.2.1 Three- Dimensional Cell Cultures 1066.2.2 Definition of Organoid 1066.2.3 Types of Organoids 1086.2.4 Applications of Organoids: Bridging In Vivo and In Vitro Studies 1086.3 Live Imaging of Organoids 1096.3.1 The Challenging Optical Properties of Organoids 1096.3.2 Selected Applications 1106.3.2.1 Widefield Microscopy 1106.3.2.2 Confocal Microscopy 1116.3.2.3 Light- Sheet Fluorescence Microscopy 1136.3.2.4 “Classical” LSFM 1146.3.2.5 Single- Objective LSFM 1156.3.2.6 Inverted (“Open- Top”) LSFM 1166.4 Conclusion: Key Points and Future Perspectives 117References 1177 Tiny Organisms, Big Advantages: Exploring Membrane Damage Responses Using Dictyostelium discoideum in High- Throughput Single- Cell Analyses 123Angélique Perret, Céline Michard, Dimitri Moreau, and Thierry Soldati7.1 Introduction 1237.2 Dictyostelium discoideum, a Workhorse of Cellular Microbiology 1257.3 Optimized Live Imaging Protocols to Track LLOMe- Mediated Endolysosomal Damage and Repair 1277.4 Perspectives/Discussion 1307.5 Annexes: Detailed Protocols 1327.5.1 Annex 1: Establishment of Stable Safe- Haven Cell Lines 1327.5.2 Annex 2: LLOMe Protocol 133References 1358 Live Imaging in Caenorhabditis elegans Neurons 141Badal Singh Chauhan and Sandhya P. Koushika8.1 Introduction 1418.2 Worm Immobilization 1418.3 Live Imaging in C. elegans 1438.3.1 Imaging Actin and Cytoskeletal Elements Associated with Actin 1438.3.2 Microtubule Imaging 1478.3.3 Calcium Imaging 1488.3.4 Mitochondria Imaging 1498.3.5 Neurotransmitter Sensors 1508.3.6 Voltage 1518.3.7 Extracellular Vesicles 1518.3.8 Retrograde Labeling of Endocytic Compartments 1528.3.9 Exocytosis/Endocytosis of Vesicles 1528.3.10 Organelle Imaging 1538.4 Conclusion 156Acknowledgments 156References 1569 It’s Just a Scratch – Lessons from Epidermal Wound Closure in Drosophila 171Dennis Klug and Sven Bogdan9.1 From Stitches, Scratches, and Cuts 1719.2 Drosophila as Model to Study Wound Healing 1729.3 Calcium Bursts Orchestrate Early Wound Signaling Across Species 1739.4 Forcing Wounds Closed – Actin Dynamics Drive Early Epidermal Wound Closure 1769.5 Tensile Forces – Junctional Remodeling in Epidermal Wound Closure 1809.6 Calcium Flashes Trigger the Cellular Immune Response by ROS Gradients 1819.7 (Wound) Closing Remarks 182Acknowledgments 182References 183  10 Intravital Imaging of Tumors 191Yookyung Jung and David Entenberg10.1 Introduction 19110.2 Capabilities of Laser Scanning Microscopy 19210.3 Essential Components of Laser Scanning Microscopes 19310.4 Challenges Inherent in Imaging Live Organisms 19410.5 Techniques for Overcoming Motion 19610.6 Unique Metrics Attainable with IVM 19710.7 Examples of Recent Applications of IVM 19910.7.1 Using Negative Contrast Imaging 19910.7.2 Intravital Imaging of Lung Metastasis Using a Permanent Lung Imaging Window 20010.7.3 Intravital Imaging of Cancer Stem Cells 20210.8 Ongoing Challenges and New Techniques to Address Them 20310.8.1 Limits to Penetration Depth 20310.8.2 Limits to the Number of Simultaneous Labels 20310.9 Conclusion 205References 205Index 211