Almost 400 years ago, Robert Hooke and Antonie van Leeuwenhoek first viewed individual cells through their early microscopes and with careful observation laid the foundation of modern cell biology. Since then, optical microscopy has been a major driving force of biological discovery. For the longest time, microscopy was limited by the inability to acquire reproducible images—not too long ago microscopic observation could only be documented by manual drawing, which requires a specialized skill set and is arguably highly subjective. Even micrography on photographic film has only limited quantitative value. Over the last two decades, however, ever-accelerating advances in optics, electronics, and digital camera technology in combination with the rise of fluorescent proteins have transformed fluorescence light microscopy from a descriptive, observational tool to a truly quantitative method.
Digital cameras are now more sensitive than the human eye, and fluorescence from individual molecules can routinely be detected. The dynamics of movement, intensity fluctuations, or distribution of fluorescently labeled structures in living cells or organisms can be measured providing important information about biological processes. This is the exciting new world of quantitative cell biology, and ideally scientific conclusions will continue to rely less and less on "representative images," but instead on quantitative analysis of digital data. Although microscopy is one of the most direct tools available to ask a scientific question, images can also be dangerously deceiving. In our modem world, the deceptive nature of images is all around us from advertising to the daily news, but in science, we must learn to objectively analyze the underlying data instead of blindly believing what we think we can see. Any digital photomicrograph is only a representation of reality that needs to be carefully scrutinized and interpreted in order to reach valid conclusions, and any analysis can only be as good as the original data.
The goals of this book are to provide the reader with a practical understanding of how digital image data are generated by modern fluorescence microscopy modalities, to outline technical and fundamental reasons that limit the accuracy and precision with which these data can be analyzed, and to provide guidance into cutting-edge technologies and image analysis that are expanding the abilities of traditional microscopy. Chapters 1-6 cover basic principles of quantitative fluorescence microscopy as well as technical aspects of objective lenses, cameras, microscope maintenance, modem live-cell imaging setups, and the properties of fluorescent proteins. Chapters 7-17 give an overview of different fluorescence microscopy techniques ranging from more established confocal imaging to state-of-the-art super-resolution strategies that circumvent the diffraction limit, and light-sheet microscopy allowing unparalleled isotropic observation of biological specimens in three dimensions. Finally, the remaining chapters describe more specific and advanced microscopy and image analysis methods. We were striving for a balanced mixture of basic information and more advanced topics, and hope that this collection will be a useful resource to anyone who wishes to venture into the brave new world of quantitative fluorescence microscopy. We would like to thank the students of the Quantitative Imaging: From Cells to Molecules course at Cold Spring Harbor Laboratory for many inspiring discussions. Last but not least, we thank all the authors who have contributed their expertise to this volume. Jennifer C. Waters Harvard Medical School; Torsten Wittmann University of California, San Francisco

Contributors xiii

Preface xix

CHAPTER 1 Concepts in Quantitative Fluorescence Microscopy 1

Jennifer C. Waters, Torsten Wittmann

1.1 Accurate and Precise Quantitation 2

1.2 Signal, Background, and Noise 3

1.3 Optical Resolution: The Point Spread Function 7

1.4 Choice of Imaging Modality 7

1.5 Sampling: Spatial and Temporal 8

1.6 Postacquisition Corrections 12

1.7 Making Compromises 15

1.8 Communicating Your Results 16

Acknowledgment 16

References 16

CHAPTER 2 Practical Considerations of Objective Lenses for Application in Cell Biology 19

Stephen T. Ross, John R. Allen, Michael W. Davidson

Introduction 20

2.1 Optical Aberrations 20

2.2 Types of Objective Lenses 22

2.3 Objective Lens Nomenclature 25

2.4 Optical Transmission and Image Intensity 25

2.5 Coverslips, Immersion Media, and Induced Aberration 27

2.6 Considerations for Specialized Techniques 31

2.7 Care and Cleaning of Optics 32

Conclusions 34

References 34

CHAPTER 3 Assessing Camera Performance for Quantitative Microscopy 35

Talley J. Lambert, Jennifer C. Waters

3.1 Introduction to Digital Cameras for Quantitative Fluorescence Microscopy 36

3.2 Camera Parameters 37

3.3 Testing Camera Performance: The Photon Transfer Curve 44

References 52

CHAPTER 4 A Practical Guide to Microscope Care and Maintenance 55

Lara J. Petrak, Jennifer C. Waters

Introduction 56

4.1 Cleaning 58

4.2 Maintenance and Testing 66

4.3 Considerations for New System Installation 74

Acknowledgments 75

References 75

CHAPTER 5 Fluorescence Live Cell Imaging 77

Andreas Ettinger, Torsten Wittmann

5.1 Fluorescence Microscopy Basics 78

5.2 The Live Cell Imaging Microscope 79

5.3 Microscope Environmental Control 83

5.4 Fluorescent Proteins 87

5.5 Other Fluorescent Probes 92

Conclusion 93

Acknowledgments 93

References 93

CHAPTER 6 Fluorescent Proteins for Quantitative Microscopy: Important Properties and Practical Evaluation 95

Nathan Christopher Shaner

6.1 Optical and Physical Properties Important for Quantitative Imaging 96

6.2 Physical Basis for Fluorescent Protein Properties 99

6.3 The Complexities of Photostability 101

6.4 Evaluation of Fluorescent Protein Performance in Vivo 106

Conclusion 108

References 109

CHAPTER 7 Quantitative Confocal Microscopy: Beyond a Pretty Picture 113

James Jonkman, Claire ML Brown, Richard W. Cole

7.1 The Classic Confocal: Blocking Out the Blur 114

7.2 You Call that Quantitative? 118

7.3 Interaction and Dynamics 123

7.4 Controls: Who Needs Them? 125

7.5 Protocols 127

Conclusions 133

References 133

CHAPTER 8 Assessing and Benchmarking Multiphoton Microscopes for Biologists 135

Kaitlin Corbin, Henry Pinkard, Sebastian Peck, Peter Beemiller, Matthew F. Krummel

Introduction: Practical Quantitative 2P Benchmarking 136

8.1 Part I: Benchmarking Inputs 136

8.2 Part II: Benchmarking Outputs 144

8.3 Troubleshooting/Optimizing 150

8.4 A Recipe for Purchasing Decisions 150

Conclusion 151

Acknowledgments 151

References 151

CHAPTER 9 Spinning-disk Confocal Microscopy: Present Technology and Future Trends 153

John Oreopoulos, Richard Berman, Mark Browne

9.1 Principle of Operation 153

9.2 Strengths and Weaknesses 155

9.3 Improvements in Light Sources 157

9.4 Improvements in Illumination 157

9.5 Improvements in Optical Sectioning and FOV 162

9.6 New Detectors 166

9.7 A Look into the Future 167

References 171

CHAPTER 10 Quantitative Deconvolution Microscopy 177

Paul C. Goodwin

Introduction 178

10.1 The Point-spread Function 180

10.2 Deconvolution Microscopy 182

10.3 Results 187

Conclusion 191

References 191

CHAPTER 11 Light Sheet Microscopy 193

Michael Weber, Michaela Mickoleit, Jan Huisken

Introduction 194

11.1 Principle of Light Sheet Microscopy 195

11.2 Implementations of Light Sheet Microscopy 198

11.3 Mounting a Specimen for Light Sheet Microscopy 203

11.4 Acquiring Data 205

11.5 Handling of Light Sheet Microscopy Data 210

References 212

CHAPTER 12 DNA Curtains: Novel Tools for Imaging Protein-Nucleic Acid Interactions at the Single-Molecule Level 217

Bridget E. Collins, Ling F. Ye, Daniel Duzdevich, Eric C. Greene

Introduction 218

12.1 Overview of TIRFM 219

12.2 Flow Cell Assembly 220

12.3 Importance of the Lipid Bilayer 221

12.4 Barriers to Lipid Diffusion 222

12.5 Different Types of DNA Curtains 223

12.6 Using DNA Curtains to Visualize Protein-DNA Interactions 226

12.7 Future Perspectives 232

Acknowledgments 232

References 232

CHAPTER 13 Nanoscale Cellular Imaging with Scanning Angle Interference Microscopy 235

Christopher DuFort, Matthew Paszek

Introduction 236

13.1 Experimental Methods and Instrumentation 241

13.2 Image Analysis and Reconstruction 250

Conclusion 250

Acknowledgments 251

References 251

CHAPTER 14 Localization Microscopy in Yeast 253

Markus Mund, Charlotte Kaplan, Jonas Ries

Introduction 254

14.1 Preparing the Yeast Strain 256

14.2 Considerations for the Choice of a Labeling Strategy 257

14.3 Preparing the Sample 260

14.4 Image Acquisition 264

14.5 Results 265

Summary 267

Acknowledgments 269

References 269

CHAPTER 15 Imaging Cellular Ultrastructure by PALM, iPALM, and Correlative iPALM-EM 273

Gleb Shtengel, Yilin Wang, Zhen Zhang, Wan Ing Goti, Harald F. Hess, Pakorn Kanchanawong

Introduction 274

15.1 Principles 275

15.2 Methods 277

15.3 Future Directions 290

Acknowledgments 291

References 292

CHAPTER 16 Seeing More with Structured Illumination Microscopy 295

Reto Fiolka

Introduction 296

16.1 Theory of Structured Illumination 297

16.2 3D SIM 302

16.3 SIM Imaging Examples 307

16.4 Practical Considerations and Potential Pitfalls 310

16.5 Discussion 311

References 312

CHAPTER 17 Structured Illumination Superresolution Imaging of the Cytoskeleton 315

Ulrike Engel

Introduction 316

17.1 Instrumentation for SIM Imaging 316

17.2 Sample Preparation 322

17.3 Minimizing Spherical Aberration 324

17.4 Multichannel SIM 327

17.5 Live Imaging with SIM 330

Acknowledgments 331

References 331

CHAPTER 18 Analysis of Focal Adhesion Turnover: A Quantitative Live-Cell Imaging Example 335

Samantha J. Stehbens, Torsten Wittmann

Introduction to Focal Adhesion Dynamics 335

18.1 FA Turnover Analysis 337

Acknowledgments 346

References 346

CHAPTER 19 Determining Absolute Protein Numbers by Quantitative Fluorescence Microscopy 347

Jolien Suzanne Verdaasdonk, Josh Lawrimore, Kerry Bloom

Introduction 348

19.1 Methods for Counting Molecules 348

19.2 Protocol foT Counting Molecules by Ratiometric Comparison of Fluorescence Intensity 356

Conclusions 361

References 361

CHAPTER 20 High-Resolution Traction Force Microscopy 367

Sergey V. Plotnikov, Benedikt Sabass, Ultich S. Schwarz, Clare M. Waterman

Introduction 368

20.1 Materials 374

20.2 Methods 381

References 392

CHAPTER 21 Experimenters' Guide to Colocalization Studies: Finding a Way Through Indicators and Quantifiers, in Practice 395

Fabrice p. Cordelieres, Susanne Bolte

Introduction 396

21.1 An Overview of Colocalization Approaches 397

Conclusion 406

References 407

CHAPTER 22 User-Friendly Tools for Quantifying the Dynamics of Cellular Morphology and Intracellular Protein Clusters 409

Denis Tsygankov, Pei-Hsuan Chu, Hsin Chen, Timothy C. Elston, Klaus M. Hariri

Introduction 410

22.1 Automated Classification of Cell Motion Types 411

22.2 GUI for Morphodynamics Classification and Ready Representation of Changes in Cell Behavior Over Time 415

22.3 Results of Morphodynamics Classification 417

22.4 Geometry-based Segmentation of Cells in Clusters 418

22.5 GUI for Cell Segmentation and Quantification of Protein Clusters 421

22.6 Results for Quantifying Protein Clusters 424

22.7 Discussion 424

Acknowledgments 426

References 426

CHAPTER 23 Ratiometric Imaging of pH Probes 429

Bree K. Grillo-HiII, Bradley A. Webb, Diane L. Barber

Introduction 430

23.1 Currently Used Ratiometric pH Probes 430

23.2 Applications 435

23.3 Protocols 438

Acknowledgments 445

References 445

CHAPTER 24 Toward Quantitative Fluorescence Microscopy with DNA Origami Nanorulers 449

Susanne Beater, Mario Raab, Philip Tinnefeld

Introduction 450

24.1 The Principle of DNA Origami 452

24.2 Functionalizing DNA Origami Structures 452

24.3 DNA Origami as Fluorescence Microscopy NanoTulers 455

24.4 Brightness References Based on DNA Origami 457

24.5 Applications of DNA Origami Nanorulers for Visualizing Resolution 458

24.6 How to Choose an Appropriate Nanoruler for a Given Application 461

References 463

CHAPTER 25 Imaging and Physically Probing Kinetochores in Live Dividing Cells 467

Jonathan Kuhn, Sophie Dumont

Introduction 468

25.1 Spindle Compression to Image and Perturb Kinetochores 469

25.2 Imaging Kinetochore Dynamics at Subpixel Resolution Via Two-Color Reporter Probes 477

Conclusion and Outlook 484

Acknowledgments 485

References 485

CHAPTER 26 Adaptive Fluorescence Microscopy by Online Feedback Image Analysis 489

Christian Tischer, Volker Hilsenstein, Kirsten Hanson, Rainer Pepperkok

Introduction 490

26.1 Requirements for Adaptive Feedback Microscopy 492

26.2 Selected Applications 493

Acknowledgments 501

References 501

CHAPTER 27 Open-Source Solutions for SPIMage Processing 505

Christopher Schmied, Evangelia Stamataki, Pavel Tomancak

Introduction 506

27.1 Prerequisites 509

27.2 Overview of the SPIM Image-Processing Pipeline 512

27.3 Bead-Based Registration 513

27.4 Multiview Fusion 518

27.5 Processing on a High-Performance Cluster 524

27.6 Future Applications 525

References 527

CHAPTER 28 Second-Harmonic Generation Imaging of Cancer 531

Adib Keikhosravi, Jeremy S. Bredfeldt, Md. Abdul Kader Sagar, Kevin W. Eliceiri

Introduction 532

28.1 SHG Physical and Chemical Background 532

28.2 SHG Instrumentation 533

28.3 Collagen Structure as a Biomarker 533

28.4 SHG in Cancer Research 535

28.5 Quantitative Analysis of SHG Images 541

Conclusion 541

References. 542

Index 547

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