Foreword by Ernst H. K. Stelzer xvii Preface xxi 1 Let There be Light Sheet 1 Pavel Tomancák and Emmanuel G. Reynaud 1.1 Historical Context of Light Sheet Microscopy - Ultramicroscopy 1 1.2 Light Sheet Imaging Across the Twentieth Century 3 1.3 And here Comes the Flood 3 1.4 The Building of a Community 6 References 8 2 Illumination in Light Sheet Fluorescence Microscopy 11 Rory M.
Power and Jan Huisken 2.1 Introduction 11 2.2 Axial Resolution and Optical Sectioning in Light Sheet Microscopy 13 2.2.1 The Point Spread Function in Fluorescence Microscopy 13 2.2.2 The Point Spread Function in Light Sheet Fluorescence Microscopy 14 2.3 Light Sheet Dimensions 16 2.
3.1 Gaussian Optics Description of Beam Focusing (x,z Axes) 18 2.3.2 Methods of Light Sheet Production (y Axis) 20 2.4 Practical Light Sheet Generation 21 2.4.1 Static and Pivoted Light Sheets 21 2.4.
2 Scanned Light Sheets 27 2.5 Degradation of the Light Sheet in Tissue 29 2.5.1 Absorption of the Light Sheet in Tissue 29 2.5.2 Refraction of the Light Sheet in Tissue 31 2.5.3 Scattering of the Light Sheet in Tissue 31 2.
6 Challenges and Benefits of Light Sheet Modes 32 2.6.1 Parallelization of the Light Sheet 32 2.6.2 Image Artifacts Arising from Light Sheet Illumination 34 2.6.3 Homogeneity of Light Sheet Illumination 35 2.6.
4 Robustness and Simplicity of Light Sheet Generation 39 2.6.5 The Merits of Static, Pivoted, and Scanned Light Sheets 39 2.7 Multiphoton Excitation 40 2.7.1 Two-Photon Light Sheets 40 2.7.2 Two-Photon Light Sheet Dimensions 41 2.
7.3 Comparison with One-Photon Light Sheet Microscopy 45 2.7.4 Comparison with Laser-Scanning Multiphoton Microscopy 47 2.8 Multi-View Illumination 48 2.9 High-Resolution Imaging 51 2.9.1 Geometric Limitations for High-Resolution Imaging 51 2.
9.1.1 Oblique Light Sheets 52 2.9.1.2 Reflected Light Sheets 52 2.9.2 Diffractive Limitations for High-Resolution Imaging 54 2.
9.2.1 Bessel Beams 54 2.9.2.2 Axially Swept Beams 59 2.9.2.
3 Photophysical Approaches 61 2.10 Conclusions 61 References 63 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 67 3.2 A Few Basic Rules 68 3.2.1 Rule 0 - Don''t Panic! Become Enthusiastic! 68 3.2.
2 Rule 1 - Keep it Clean 68 3.2.3 Rule 2 - The Light Comes Sideways 69 3.2.4 Rule 3 - The Theory does not Apply to your Sample 71 3.2.5 Rule 4 - Sample Geometry Matters 71 3.2.
6 Rule 5 - Know Your System Well 72 3.2.7 Rule 6 - How Does Your Sample Move? 73 3.2.8 Rule 7 - What Was Lost? 73 3.2.9 Rule 8 - Consistency is Key 74 3.3 The Light-Matter Conundrum 74 3.
4 Hydrogels 75 3.4.1 Preparation 76 3.5 Glues 77 3.6 Sample Holders 77 3.7 Clearing 79 3.8 Cleaning, Labelling, and Storing Samples 83 3.9 An Example: Time-lapse Live Imaging of Three-dimensional Cultures 84 3.
9.1 Environmental Control: Temperature, pH, Oxygenation 84 3.9.2 Perfusion-based Environmental Control 88 3.9.3 Sample Holders for the Live Imaging of Three-dimensional Cell Cultures 91 3.9.3.
1 Agarose Beakers 91 3.9.3.2 FEP-foil Sample Holders 91 3.9.4 References 93 3.10 A Bit of Literature 94 3.10.
1 Reference Guides 95 3.10.2 Your Favorite Models 95 3.10.3 Others 96 3.10.4 Protocol Videos 97 Bibliography 98 4 Detection in a Light Sheet Microscope 101 Jacob Licea-Rodriguez, Omar E. Olarte, Jordi Andilla, and Pablo Loza-Alvarez 4.
1 Introduction 101 4.2 Image Formation in LSFM 102 4.2.1 WFM Scheme 102 4.2.2 LSFM Scheme 104 4.2.3 Practical Design Examples of an LSFM 106 4.
3 Advanced Detection Schemes 108 4.3.1 Spectrally Resolved 108 4.3.2 Contrast Enhancement (Confocal Line) 113 4.3.3 Aberration Correction (Adaptive Optics) 115 4.3.
4 Fast Volumetric Imaging 116 4.3.4.1 Inverted SPIM 117 4.3.4.2 Remote Focusing Using Tunable Lens 118 4.3.
4.3 Opm-scape 119 4.3.4.4 Wavefront Coding 120 4.4 Conclusions 120 References 121 5 Light Sheet Microscope Configurations 125 Michael Weber and Emilio J. Gualda 5.1 LSFM Architectures 125 5.
1.1 Multiple Objective Lens Configurations 125 5.1.2 Single Objective Lens Configurations 126 5.1.3 Opposing Objective Lens Configurations 128 5.2 Recording Three-Dimensional Image Data 128 5.2.
1 Moving the Sample 128 5.2.2 Moving Detection and Illumination 130 5.3 Configurations that Expand on Specific Capabilities 131 5.3.1 First Light Sheet: Increasing Sample Viability 131 5.3.2 Imaging Easier: Increasing Flexibility 132 5.
3.3 Imaging Deeper: Increasing Penetration Depth 132 5.3.4 Imaging Wider: Increasing the Effective Field of View 133 5.3.5 Imaging All Around: Increasing the Isotropy 134 5.3.6 Imaging Brighter: Increasing Contrast 135 5.
3.7 Imaging Faster in 3D: Increasing Volumetric Temporal Resolution 136 5.3.8 Imaging Bigger: Increasing Sample Volume 137 5.3.9 Imaging Smaller: Increasing Spatial Resolution 138 5.3.10 Imaging More: Increasing Throughput 140 5.
4 Summary 142 References 142 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 Tomancák, Wiebke Jahr, Benjamin Schmid, Jan Huisken, and Emmanuel G. Reynaud 6.1 Introduction 149 6.2 Commercial Systems 151 6.2.1 Carl Zeiss Lightsheet Z.1: Market Introduction and Experiences 151 6.
2.1.1 Introduction 151 6.2.2 ALPHA 3 : Light Sheet Fluorescence Microscope 154 6.2.2.1 Digital Light Sheet Generator 154 6.
2.2.2 Modular and Flexible Light Sheet Setup 155 6.2.3 Illumination Unit(s) 156 6.2.3.1 Sample Chamber and Holders 156 6.
2.3.2 Detection Unit 156 6.2.3.3 Software 157 6.2.3.
4 High-Speed 3D Acquisition 157 6.2.3.5 Applications 158 6.2.3.6 Summary 158 6.2.
4 Leica TCS SP8 DLS: Turning Light Sheet Microscopy Vertically 158 6.2.4.1 Light Path 159 6.2.4.2 Sample Preparation for the Leica TCS SP8 DLS 160 6.2.
4.3 Convenient Software Tools to Manage Large Data Amounts 161 6.2.4.4 Technical Specifications 163 6.2.4.5 Applications 164 6.
2.4.6 Imaging with Low Light Intensities 164 6.2.4.7 Imaging of Cleared Tissue 164 6.2.4.
8 Imaging of Fast Dynamic Processes 164 6.2.4.9 High Throughput by Multiposition Experiments and Imaging of Larger Specimens 164 6.2.4.10 Advanced Applications by Combined Imaging Methods 165 6.2.
4.11 Summary 165 6.2.5 The Large Selective Plane Illuminator (L-SPI): A Versatile Illumination Module for Large Photosensitive Samples 165 6.2.5.1 Introduction 165 6.2.
5.2 Design 166 6.2.5.3 Light Sheet Properties and Resolution 169 6.2.5.4 Sample Preparation 169 6.
2.5.5 Application 1: Fluorescence Imaging in Live Coral Samples 171 6.2.5.6 Application 2: Fluorescence Imaging in Other Live and Fixed Samples 173 6.2.5.
7 Application 3: Reflectance Imaging 173 6.2.5.8 Software, Image Processing, and Data Management 174 6.2.5.9 Price Range 175 6.2.
5.10 Acknowledgment 175 6.2.6 LUXENDO''s Modular Light Sheet Solutions Adapt Specifically to a Broad Spectrum of Diverse Samples and Applications 175 6.2.6.1 Introduction 176 6.2.
6.2 Multiple View Selective Plane Illumination Microscope (MuVi Spim) 178 6.2.6.3 Clearing 179 6.2.6.4 Inverted View Selective Plane Illumination Microscope (InVi Spim) 180 6.
2.6.5 Quantitative View Selective Plane Illumination Microscope (QuVi Spim) 182 6.2.6.6 Conclusion 183 6.2.6.
7 Acknowledgments 183 6.3 Open-Source Systems 183 6.3.1 OpenSPIM: The Do It Yourself (DIY) Selective Plane Illumination Microscopy (SPIM) Approach 183 6.3.1.1 Introduction 183 6.3.
1.2 The Principle of DIY SPIM 184 6.3.1.3 Of the Diversity of Biological Applications Using DIY SPIM Microscopy 187 6.3.1.4 Community 189 6.
3.2 eduSPIM: Light Sheet Fluorescence Microscopy in the Museum 189 6.3.2.1 Introduction 189 6.3.2.2 Optical Design 192 6.
3.2.3 Control Software and User Interface 194 6.3.2.4 Sample Choice and Sample Mounting 195 6.3.2.
5 Outreach and Discussion 196 6.3.2.6 Acknowledgments 197 References 197 Further Reading 200 Publications with Lightsheet Z. 1 200 7 Image Processing and Analysis of Light Sheet Microscopy Data 203 Akanksha Jain, Vladimir Ulman, Michal Krumnikl, Tobias Pietzsch, Stephan Preibisch, and Pavel Tomancák 7.1 Introduction 203 7.2 Multi-view SPIM Reconstruction 204 7.2.
1 Multi-view Registration 206 7.2.2 Multi-view Fusion 208 7.3 Processing of Data from Other Light Sheet Microscopy Implementations 211 7.4 Big Image Data Management and Visualization 212 7.4.1 Hierarchical Data Format 212 7.4.
2 Parallel Processing 214 7.4.3 Big Data Visualization 216 7.5 Analysis of Light Sheet Microscopy Datasets 218 7.5.1 Image Dimensional.