An indispensable guide to a novel, revolutionary fluorescence microscopy technique!
Light sheet fluorescence microscopy has revolutionized microscopy, since it allows scientists to perform experiments in an entirely different manner and to record data that had not been accessible before. With contributions from noted experts in the fields of physics, biology, and computer science, Light Sheet Fluorescence Microscopy is a unique guide that offers a practical approach to the subject, including information on the basics of light sheet fluorescence microscopy, instrumentation, applications, sample preparation, and data analysis.
Comprehensive in scope, the book is filled with the cutting-edge methods as well as valuable insider tips. Grounded in real-world applications, the book includes chapters from major manufacturers that explores their recent systems and developments. In addition, the book hightlights a discussion of a “do-it-yourself” light sheet microscope, making the technique affordable for every laboratory.
This important textbook: - Serves as an easy-to-understand introduction to light sheet-based fluorescence - Includes numerous tips and tricks for advanced practitioners - Provides in-depth information on hardware and software solutions for a straightforward implementation of light sheet fluorescence microscopy in the lab - Includes chapters from the major manufacturers including Zeiss, Leica, Lavision Biotech, Phase View, and Asi
Aimed at cell biologists, biophysicists, developmental biologists, and neuro-biologists, Light Sheet Fluorescence Microscopy offers a comprehensive overview of the most recent applications of this microscopy technique.
Table of Contents
Foreword by Ernst H. K. Stelzer xvii
Preface xxi
1 Let There be Light Sheet 1
Pavel Tomančák 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 Tomančák, 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 Tomančák
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 Dimensionality Reduction for Better Analysis 219
7.5.2 Segmentation and Tracking in Light Sheet Data 219
7.5.3 Atlas Registration 222
7.6 Conclusion 223
References 223
8 Imaging Molecular Dynamics Using a Light Sheet Microscope 231
Jagadish Sankaran and Thorsten Wohland
8.1 Introduction 231
8.2 Fluorescence Techniques Using Light Sheet Illumination 232
8.2.1 Fluorescence Correlation Spectroscopy 232
8.2.2 Fluorescence Recovery After Photobleaching 235
8.2.3 Single-Particle Tracking 236
8.2.4 Förster Resonance Energy Transfer 238
8.2.5 Fluorescence Anisotropy Imaging 240
8.2.6 Fluorescence Lifetime Imaging Microscopy 241
8.3 Instrumentation 243
8.3.1 Light Sheet Microscope Configurations 243
8.3.2 Objectives and Cameras 247
8.4 Considerations for Acquisition Parameters 249
8.4.1 Light Sheet Thickness Versus Field of View 249
8.4.2 Field of View Versus Frame Rate 250
8.4.3 Pixel Size Versus Spatial Resolution 251
8.4.4 Pixel Size Versus Field of View and Frame Rate 251
8.4.5 Data Rate 251
8.4.6 Synchronous Versus Asynchronous Read-Out 251
8.4.7 Photobleaching and Phototoxicity 252
8.5 Applications of Fluorescence Techniques Performed Using Light Sheet Microscopes 254
8.5.1 Fluorescence Correlation Spectroscopy 254
8.5.2 Fluorescence Recovery After Photobleaching 255
8.5.3 Single-Particle Tracking 256
8.5.4 Förster Resonance Energy Transfer 257
8.5.5 Fluorescence Anisotropy Imaging 257
8.5.6 Fluorescence Lifetime Imaging Microscopy 258
8.6 Concluding Remarks 258
References 260
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 269
9.2 3D Imaging of Rare Cells/Events 274
9.2.1 Immunology 274
9.2.2 Cryptococci Infections 276
9.3 Full 3D Imaging and Analysis for Diagnostics 279
9.3.1 Alzheimer’s Disease 279
9.3.2 Toward Preclinical Diagnosis Through LSFM Cancer Imaging: Shedding Light on Whole Tumors 281
9.3.2.1 Angiogenesis 282
9.3.2.2 Metastatic Invasion 285
9.4 Population-Based Analysis in Mouse Brains: Toward a Systems Perspective 286
9.4.1 Cytoarchitectonic Variation in Neuronal Circuits 288
9.4.2 Cell and Dendritic Density Mapping 289
9.4.3 Voxel-Based Morphometry in Dendritic Density Maps Recapitulates Single-Neuron Dendritic Alterations 290
9.4.4 Development of Computational Tools for Generative Modeling of 3D Neuronal Circuits 293
9.5 4D Imaging of Plant Development 295
9.5.1 Challenges of Live Imaging of Plants 295
9.5.2 Key Elements for LSFM Live Imaging of Plants 295
9.5.3 In toto Time Resolved Imaging of Plants 297
9.6 Perspectives on Whole-Organ Imaging: What’s Next? 298
9.a Appendix: Challenges and Insights into Image Analysis Workflows for Large Volumes of LSFM Bioimage Data 299
9.a.1 Automated Image Analysis in LSFM Applications: Challenges 299
9.a.2 Automated Image Analysis: Applications 300
9.a.3 Automated Image Analysis: Strategies to Reduce Computational Complexity 302
9.a.3.1 Process Sub-volumes from Selected Regions 302
9.a.3.2 Full Sample Brick Splitting 302
9.a.3.3 Full Sample 2.5D Analysis 302
9.a.4 Strategies to Improve Image Analysis Flexibility and Accuracy 302
9.a.4.1 Machine Learning Algorithms 302
9.a.4.2 Result Montage and Linking to Target Positions 303
9.a.4.3 Intelligent Microscopy 303
Acknowledgments 304
References 305
10 Single-Objective Light-Sheet Microscopy 317
Venkatakaushik Voleti and Elizabeth M. C. Hillman
10.1 Introduction: Why Use Single-Objective Systems? 317
10.2 Optical Configurations and Design Considerations for Single-Objective Light Sheet 319
10.2.1 Optical Layouts 319
10.2.1.1 Horizontal-Sheet Single-Objective Systems 320
10.2.1.2 Oblique- and Axial-Illumination Single-Objective Systems 320
10.2.1.3 HILO and VAEM Single-Objective Methods 321
10.2.2 Excitation Side: Light-Sheet Formation and Parameters 322
10.2.2.1 Excitation Configurations for Horizontal-Sheet Single-Objective Systems 323
10.2.2.2 Excitation Configurations for Oblique Sheet Single-Objective Systems 323
10.2.2.3 Beam-Waist Considerations for Oblique Versus Horizontal-Sheet Configurations 324
10.2.2.4 Advanced Methods for Excitation Sheet Formation 324
10.2.3 Detection Optics: Image Formation and Rotation 324
10.2.3.1 Detection-Side Optics for Horizontal-Sheet Systems 324
10.2.3.2 Detection-Side Strategies for Single-Objective Oblique and Axial Light-Sheet Systems 325
10.2.3.3 Camera Field of View Considerations for Oblique and Axial Sheet Systems 327
10.2.4 Scanning Approaches for Volumetric Imaging 328
10.2.4.1 Volumetric Image Formation in Horizontal-Sheet Single-Objective Systems 328
10.2.4.2 Volumetric Image Formation Using Stage Scanning 329
10.2.4.3 Volumetric Scanning in Oblique Illumination Systems 330
10.2.5 Factors and Trade-Offs Affecting Imaging Performance 331
10.2.5.1 Factors Affecting Penetration Depth 331
10.2.5.2 Factors Affecting Field of View, Resolution Homogeneity, and Isotropy 332
10.2.5.3 Comparing Single-Objective Light-Sheet Methods with Confocal Microscopy 332
10.2.6 Image Processing, Display, and Analysis 334
10.3 Applications 335
10.3.1 Super-Resolution Imaging with Single-Objective Light-Sheet Geometries 335
10.3.2 Large Field of View (FOV), High-Throughput Imaging with Oblique Light-Sheet Systems 336
10.3.3 High-Speed Functional Imaging of Brain Activity Using SCAPE 337
10.4 Conclusions 338
Acknowledgments 339
Conflict of Interest 340
References 340
11 HowtoOrganizeaPracticalCourseonLightSheet Microscopy 345
Emmanuel G. Reynaud, Jan Peychl, and Pavel Tomančák
11.1 Introduction 345
11.2 General Course Set-up 345
11.3 Samples, Samples, Samples 349
11.4 Light Sheet Iron 352
11.5 Late Night Data Processing and Analysis 355
11.6 Trying Not to Drown in the Data 357
11.7 How to Create a Great Course Atmosphere 360
References 362
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 365
12.2 Profile of User Base 365
12.2.1 User Rules 366
12.2.1.1 Weekly Schedule (Example from Z.1 System) 366
12.2.1.2 Storage Space 366
12.2.1.3 User Mailing List 366
12.2.2 General Protocol 367
12.2.2.1 General attitude at the system (Z.1, Zeiss) 367
12.3 Applications 367
12.3.1 Live Cell Microscopy 367
12.3.2 Multiview Imaging of Fixed, Cleared Biological Samples 369
12.3.3 Material Science, Tissue Engineering 369
12.3.4 Hybrid Techniques 370
12.3.5 Helping Projects Where Light Sheet Is Not the Answer 371
12.4 Data and IT Aspects of LSFMs in a Facility 371
12.4.1 MPI-CBG Light Sheet Data Experience 372
12.4.2 LSFM - Hardware for Data Handling 373
12.4.2.1 Data Transfer: Faster Internal MPI-CBG Network (10 GB/s) 374
12.4.2.2 Hardware for Data Processing, Storage, and Archival 375
12.4.3 LSFM - Image Processing Software Solutions 378
12.4.4 Data and Users 379
12.4.4.1 Big Data Awareness: User Education 379
12.5 Past and Outlook 379
12.6 Conclusion 379
References 380
Index 383
