1 Let There be Light Sheet 1 Pavel Tomančák and Emmanuel G. Reynaud -- 1.1 Historical Context of Light Sheet Microscopy - Ultramicroscopy -- 1.2 Light Sheet Imaging Across the Twentieth Century -- 1.3 And here Comes the Flood -- 1.4 The Building of a Community -- References -- 2 Illumination in Light Sheet Fluorescence Microscopy 11 Rory M. Power and Jan Huisken -- 2.1 Introduction -- 2.2 Axial Resolution and Optical Sectioning in Light Sheet Microscopy -- 2.2.1 The Point Spread Function in Fluorescence Microscopy -- 2.2.2 The Point Spread Function in Light Sheet Fluorescence Microscopy -- 2.3 Light Sheet Dimensions -- 2.3.1 Gaussian Optics Description of Beam Focusing (x,z Axes) -- 2.3.2 Methods of Light Sheet Production (y Axis) -- 2.4 Practical Light Sheet Generation -- 2.4.1 Static and Pivoted Light Sheets -- 2.4.2 Scanned Light Sheets -- 2.5 Degradation of the Light Sheet in Tissue -- 2.5.1 Absorption of the Light Sheet in Tissue -- 2.5.2 Refraction of the Light Sheet in Tissue -- 2.5.3 Scattering of the Light Sheet in Tissue -- 2.6 Challenges and Benefits of Light Sheet Modes -- 2.6.1 Parallelization of the Light Sheet -- 2.6.2 Image Artifacts Arising from Light Sheet Illumination -- 2.6.3 Homogeneity of Light Sheet Illumination -- 2.6.4 Robustness and Simplicity of Light Sheet Generation -- 2.6.5 The Merits of Static, Pivoted, and Scanned Light Sheets -- 2.7 Multiphoton Excitation -- 2.7.1 Two-Photon Light Sheets -- 2.7.2 Two-Photon Light Sheet Dimensions -- 2.7.3 Comparison with One-Photon Light Sheet Microscopy -- 2.7.4 Comparison with Laser-Scanning Multiphoton Microscopy -- 2.8 Multi-View Illumination -- 2.9 High-Resolution Imaging -- 2.9.1 Geometric Limitations for High-Resolution Imaging -- 2.9.1.1 Oblique Light Sheets -- 2.9.1.2 Reflected Light Sheets -- 2.9.2 Diffractive Limitations for High-Resolution Imaging -- 2.9.2.1 Bessel Beams -- 2.9.2.2 Axially Swept Beams -- 2.9.2.3 Photophysical Approaches -- 2.10 Conclusions -- References -- 3 A Small Guide on How to Mount a Sample in a Light-Sheet Microscope 67 Francesco Pampaloni, Edward Lachica, Jacques Paysan, and Emmanuel G. Reynaud -- 3.1 Introduction -- 3.2 A Few Basic Rules -- 3.2.1 Rule 0 - Don't Panic! Become Enthusiastic! -- 3.2.2 Rule 1 - Keep it Clean -- 3.2.3 Rule 2 - The Light Comes Sideways -- 3.2.4 Rule 3 - The Theory does not Apply to your Sample -- 3.2.5 Rule 4 - Sample Geometry Matters -- 3.2.6 Rule 5 - Know Your System Well -- 3.2.7 Rule 6 - How Does Your Sample Move? -- 3.2.8 Rule 7 - What Was Lost? -- 3.2.9 Rule 8 - Consistency is Key -- 3.3 The Light-Matter Conundrum -- 3.4 Hydrogels -- 3.4.1 Preparation -- 3.5 Glues -- 3.6 Sample Holders -- 3.7 Clearing -- 3.8 Cleaning, Labelling, and Storing Samples -- 3.9 An Example: Time-lapse Live Imaging of Three-dimensional Cultures -- 3.9.1 Environmental Control: Temperature, pH, Oxygenation -- 3.9.2 Perfusion-based Environmental Control -- 3.9.3 Sample Holders for the Live Imaging of Three-dimensional Cell Cultures -- 3.9.3.1 Agarose Beakers -- 3.9.3.2 FEP-foil Sample Holders -- 3.9.4 References -- 3.10 A Bit of Literature -- 3.10.1 Reference Guides -- 3.10.2 Your Favorite Models -- 3.10.3 Others -- 3.10.4 Protocol Videos -- Bibliography -- 4 Detection in a Light Sheet Microscope 101 Jacob Licea-Rodriguez, Omar E. Olarte, Jordi Andilla, and Pablo Loza-Alvarez -- 4.1 Introduction -- 4.2 Image Formation in LSFM -- 4.2.1 WFM Scheme -- 4.2.2 LSFM Scheme -- 4.2.3 Practical Design Examples of an LSFM -- 4.3 Advanced Detection Schemes -- 4.3.1 Spectrally Resolved -- 4.3.2 Contrast Enhancement (Confocal Line) -- 4.3.3 Aberration Correction (Adaptive Optics) -- 4.3.4 Fast Volumetric Imaging -- 4.3.4.1 Inverted SPIM -- 4.3.4.2 Remote Focusing Using Tunable Lens -- 4.3.4.3 Opm-scape -- 4.3.4.4 Wavefront Coding -- 4.4 Conclusions -- References -- 5 Light Sheet Microscope Configurations 125 Michael Weber and Emilio J. Gualda -- 5.1 LSFM Architectures -- 5.1.1 Multiple Objective Lens Configurations -- 5.1.2 Single Objective Lens Configurations -- 5.1.3 Opposing Objective Lens Configurations -- 5.2 Recording Three-Dimensional Image Data -- 5.2.1 Moving the Sample -- 5.2.2 Moving Detection and Illumination -- 5.3 Configurations that Expand on Specific Capabilities -- 5.3.1 First Light Sheet: Increasing Sample Viability -- 5.3.2 Imaging Easier: Increasing Flexibility -- 5.3.3 Imaging Deeper: Increasing Penetration Depth -- 5.3.4 Imaging Wider: Increasing the Effective Field of View -- 5.3.5 Imaging All Around: Increasing the Isotropy -- 5.3.6 Imaging Brighter: Increasing Contrast -- 5.3.7 Imaging Faster in 3D: Increasing Volumetric Temporal Resolution -- 5.3.8 Imaging Bigger: Increasing Sample Volume -- 5.3.9 Imaging Smaller: Increasing Spatial Resolution -- 5.3.10 Imaging More: Increasing Throughput -- 5.4 Summary -- References -- 6 Commercial and Open-Source Systems 149 Annette Bergter, Helmut Lippert, Gael Launay, Petra Haas, Isabelle Koester, Pierre P. Laissue, Tomas Parrado, Jeremy Graham, Jürgen Mayer, Girstmair Johannes, Pavel Tomančák, Wiebke Jahr, Benjamin Schmid, Jan Huisken, and Emmanuel G. Reynaud -- 6.1 Introduction -- 6.2 Commercial Systems -- 6.2.1 Carl Zeiss Lightsheet Z.1: Market Introduction and Experiences -- 6.2.1.1 Introduction -- 6.2.2 ALPHA 3 : Light Sheet Fluorescence Microscope -- 6.2.2.1 Digital Light Sheet Generator -- 6.2.2.2 Modular and Flexible Light Sheet Setup -- 6.2.3 Illumination Unit(s) -- 6.2.3.1 Sample Chamber and Holders -- 6.2.3.2 Detection Unit -- 6.2.3.3 Software -- 6.2.3.4 High-Speed 3D Acquisition -- 6.2.3.5 Applications -- 6.2.3.6 Summary -- 6.2.4 Leica TCS SP8 DLS: Turning Light Sheet Microscopy Vertically -- 6.2.4.1 Light Path -- 6.2.4.2 Sample Preparation for the Leica TCS SP8 DLS -- 6.2.4.3 Convenient Software Tools to Manage Large Data Amounts -- 6.2.4.4 Technical Specifications -- 6.2.4.5 Applications -- 6.2.4.6 Imaging with Low Light Intensities -- 6.2.4.7 Imaging of Cleared Tissue -- 6.2.4.8 Imaging of Fast Dynamic Processes -- 6.2.4.9 High Throughput by Multiposition Experiments and Imaging of Larger Specimens -- 6.2.4.10 Advanced Applications by Combined Imaging Methods -- 6.2.4.11 Summary -- 6.2.5 The Large Selective Plane Illuminator (L-SPI): A Versatile Illumination Module for Large Photosensitive Samples -- 6.2.5.1 Introduction -- 6.2.5.2 Design -- 6.2.5.3 Light Sheet Properties and Resolution -- 6.2.5.4 Sample Preparation -- 6.2.5.5 Application 1: Fluorescence Imaging in Live Coral Samples -- 6.2.5.6 Application 2: Fluorescence Imaging in Other Live and Fixed Samples -- 6.2.5.7 Application 3: Reflectance Imaging -- 6.2.5.8 Software, Image Processing, and Data Management -- 6.2.5.9 Price Range -- 6.2.5.10 Acknowledgment -- 6.2.6 LUXENDO's Modular Light Sheet Solutions Adapt Specifically to a Broad Spectrum of Diverse Samples and Applications -- 6.2.6.1 Introduction -- 6.2.6.2 Multiple View Selective Plane Illumination Microscope (MuVi Spim) -- 6.2.6.3 Clearing -- 6.2.6.4 Inverted View Selective Plane Illumination Microscope (InVi Spim) -- 6.2.6.5 Quantitative View Selective Plane Illumination Microscope (QuVi Spim) -- 6.2.6.6 Conclusion -- 6.2.6.7 Acknowledgments -- 6.3 Open-Source Systems -- 6.3.1 OpenSPIM: The Do It Yourself (DIY) Selective Plane Illumination Microscopy (SPIM) Approach -- 6.3.1.1 Introduction -- 6.3.1.2 The Principle of DIY SPIM -- 6.3.1.3 Of the Diversity of Biological Applications Using DIY SPIM Microscopy -- 6.3.1.4 Community -- 6.3.2 eduSPIM: Light Sheet Fluorescence Microscopy in the Museum -- 6.3.2.1 Introduction -- 6.3.2.2 Optical Design -- 6.3.2.3 Control Software and User Interface -- 6.3.2.4 Sample Choice and Sample Mounting -- 6.3.2.5 Outreach and Discussion -- 6.3.2.6 Acknowledgments -- References -- Further Reading -- Publications with Lightsheet Z. 1 -- 7 Image Processing and Analysis of Light Sheet Microscopy Data 203 Akanksha Jain, Vladimir Ulman, Michal Krumnikl, Tobias Pietzsch, Stephan Preibisch, and Pavel Tomančák -- 7.1 Introduction -- 7.2 Multi-view SPIM Reconstruction -- 7.2.1 Multi-view Registration -- 7.2.2 Multi-view Fusion -- 7.3 Processing of Data from Other Light Sheet Microscopy Implementations -- 7.4 Big Image Data Management and Visualization -- 7.4.1 Hierarchical Data Format -- 7.4.2 Parallel Processing -- 7.4.3 Big Data Visualization -- 7.5 Analysis of Light Sheet Microscopy Datasets -- 7.5.1 Image Dimensio.

nality Reduction for Better Analysis -- 7.5.2 Segmentation and Tracking in Light Sheet Data -- 7.5.3 Atlas Registration -- 7.6 Conclusion -- References -- 8 Imaging Molecular Dynamics Using a Light Sheet Microscope 231 Jagadish Sankaran and Thorsten Wohland -- 8.1 Introduction -- 8.2 Fluorescence Techniques Using Light Sheet Illumination -- 8.2.1 Fluorescence Correlation Spectroscopy -- 8.2.2 Fluorescence Recovery After Photobleaching -- 8.2.3 Single-Particle Tracking -- 8.2.4 Förster Resonance Energy Transfer -- 8.2.5 Fluorescence Anisotropy Imaging -- 8.2.6 Fluorescence Lifetime Imaging Microscopy -- 8.3 Instrumentation -- 8.3.1 Light Sheet Microscope Configurations -- 8.3.2 Objectives and Cameras -- 8.4 Considerations for Acquisition Parameters -- 8.4.1 Light Sheet Thickness Versus Field of View -- 8.4.2 Field of View Versus Frame Rate -- 8.4.3 Pixel Size Versus Spatial Resolution -- 8.4.4 Pixel Size Versus Field of View and Frame Rate -- 8.4.5 Data Rate -- 8.4.6 Synchronous Versus Asynchronous Read-Out -- 8.4.7 Photobleaching and Phototoxicity -- 8.5 Applications of Fluorescence Techniques Performed Using Light Sheet Microscopes -- 8.5.1 Fluorescence Correlation Spectroscopy -- 8.5.2 Fluorescence Recovery After Photobleaching -- 8.5.3 Single-Particle Tracking -- 8.5.4 Förster Resonance Energy Transfer -- 8.5.5 Fluorescence Anisotropy Imaging -- 8.5.6 Fluorescence Lifetime Imaging Microscopy -- 8.6 Concluding Remarks -- References -- 9 Light-Sheet Applications: From Rare Cell Detection to Full Organ Analysis 269 Julien Colombelli, Sébastien Tosi, Alexis Maizel, Linus Manubens Gil, and Jim Swoger -- 9.1 Introduction -- 9.2 3D Imaging of Rare Cells/Events -- 9.2.1 Immunology -- 9.2.2 Cryptococci Infections -- 9.3 Full 3D Imaging and Analysis for Diagnostics -- 9.3.1 Alzheimer's Disease -- 9.3.2 Toward Preclinical Diagnosis Through LSFM Cancer Imaging: Shedding Light on Whole Tumors -- 9.3.2.1 Angiogenesis -- 9.3.2.2 Metastatic Invasion -- 9.4 Population-Based Analysis in Mouse Brains: Toward a Systems Perspective -- 9.4.1 Cytoarchitectonic Variation in Neuronal Circuits -- 9.4.2 Cell and Dendritic Density Mapping -- 9.4.3 Voxel-Based Morphometry in Dendritic Density Maps Recapitulates Single-Neuron Dendritic Alterations -- 9.4.4 Development of Computational Tools for Generative Modeling of 3D Neuronal Circuits -- 9.5 4D Imaging of Plant Development -- 9.5.1 Challenges of Live Imaging of Plants -- 9.5.2 Key Elements for LSFM Live Imaging of Plants -- 9.5.3 In toto Time Resolved Imaging of Plants -- 9.6 Perspectives on Whole-Organ Imaging: What's Next? -- 9.a Appendix: Challenges and Insights into Image Analysis Workflows for Large Volumes of LSFM Bioimage Data -- 9.a.1 Automated Image Analysis in LSFM Applications: Challenges -- 9.a.2 Automated Image Analysis: Applications -- 9.a.3 Automated Image Analysis: Strategies to Reduce Computational Complexity -- 9.a.3.1 Process Sub-volumes from Selected Regions -- 9.a.3.2 Full Sample Brick Splitting -- 9.a.3.3 Full Sample 2.5D Analysis -- 9.a.4 Strategies to Improve Image Analysis Flexibility and Accuracy -- 9.a.4.1 Machine Learning Algorithms -- 9.a.4.2 Result Montage and Linking to Target Positions -- 9.a.4.3 Intelligent Microscopy -- Acknowledgments -- References -- 10 Single-Objective Light-Sheet Microscopy 317 Venkatakaushik Voleti and Elizabeth M. C. Hillman -- 10.1 Introduction: Why Use Single-Objective Systems? -- 10.2 Optical Configurations and Design Considerations for Single-Objective Light Sheet -- 10.2.1 Optical Layouts -- 10.2.1.1 Horizontal-Sheet Single-Objective Systems -- 10.2.1.2 Oblique- and Axial-Illumination Single-Objective Systems -- 10.2.1.3 HILO and VAEM Single-Objective Methods -- 10.2.2 Excitation Side: Light-Sheet Formation and Parameters -- 10.2.2.1 Excitation Configurations for Horizontal-Sheet Single-Objective Systems -- 10.2.2.2 Excitation Configurations for Oblique Sheet Single-Objective Systems -- 10.2.2.3 Beam-Waist Considerations for Oblique Versus Horizontal-Sheet Configurations -- 10.2.2.4 Advanced Methods for Excitation Sheet Formation -- 10.2.3 Detection Optics: Image Formation and Rotation -- 10.2.3.1 Detection-Side Optics for Horizontal-Sheet Systems -- 10.2.3.2 Detection-Side Strategies for Single-Objective Oblique and Axial Light-Sheet Systems -- 10.2.3.3 Camera Field of View Considerations for Oblique and Axial Sheet Systems -- 10.2.4 Scanning Approaches for Volumetric Imaging -- 10.2.4.1 Volumetric Image Formation in Horizontal-Sheet Single-Objective Systems -- 10.2.4.2 Volumetric Image Formation Using Stage Scanning -- 10.2.4.3 Volumetric Scanning in Oblique Illumination Systems -- 10.2.5 Factors and Trade-Offs Affecting Imaging Performance -- 10.2.5.1 Factors Affecting Penetration Depth -- 10.2.5.2 Factors Affecting Field of View, Resolution Homogeneity, and Isotropy -- 10.2.5.3 Comparing Single-Objective Light-Sheet Methods with Confocal Microscopy -- 10.2.6 Image Processing, Display, and Analysis -- 10.3 Applications -- 10.3.1 Super-Resolution Imaging with Single-Objective Light-Sheet Geometries -- 10.3.2 Large Field of View (FOV), High-Throughput Imaging with Oblique Light-Sheet Systems -- 10.3.3 High-Speed Functional Imaging of Brain Activity Using SCAPE -- 10.4 Conclusions -- Acknowledgments -- Conflict of Interest -- References -- 11 HowtoOrganizeaPracticalCourseonLightSheet Microscopy 345 Emmanuel G. Reynaud, Jan Peychl, and Pavel Tomančák -- 11.1 Introduction -- 11.2 General Course Set-up -- 11.3 Samples, Samples, Samples -- 11.4 Light Sheet Iron -- 11.5 Late Night Data Processing and Analysis -- 11.6 Trying Not to Drown in the Data -- 11.7 How to Create a Great Course Atmosphere -- References -- 12 Light-Sheet Microscopy Technology in the Multiuser Environment of a Core Imaging Facility - Practical Considerations 365 M. Marcello, D. Accardi, S. Bundshuch, J. Oegema, A. Andreev, Emmanuel G. Reynaud, and Jan Peychl -- 12.1 Introduction -- 12.2 Profile of User Base -- 12.2.1 User Rules -- 12.2.1.1 Weekly Schedule (Example from Z.1 System) -- 12.2.1.2 Storage Space -- 12.2.1.3 User Mailing List -- 12.2.2 General Protocol -- 12.2.2.1 General attitude at the system (Z.1, Zeiss) -- 12.3 Applications -- 12.3.1 Live Cell Microscopy -- 12.3.2 Multiview Imaging of Fixed, Cleared Biological Samples -- 12.3.3 Material Science, Tissue Engineering -- 12.3.4 Hybrid Techniques -- 12.3.5 Helping Projects Where Light Sheet Is Not the Answer -- 12.4 Data and IT Aspects of LSFMs in a Facility -- 12.4.1 MPI-CBG Light Sheet Data Experience -- 12.4.2 LSFM - Hardware for Data Handling -- 12.4.2.1 Data Transfer: Faster Internal MPI-CBG Network (10 GB/s) -- 12.4.2.2 Hardware for Data Processing, Storage, and Archival -- 12.4.3 LSFM - Image Processing Software Solutions -- 12.4.4 Data and Users -- 12.4.4.1 Big Data Awareness: User Education -- 12.5 Past and Outlook -- 12.6 Conclusion -- References -- Index.