Quantitative Imaging in Cell Biology

1st Edition, Volume 123 - June 20, 2014
Editors: Jennifer Waters, Torsten Wittmann
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 4 2 0 1 3 8 - 5
eBook ISBN:
9 7 8 - 0 - 1 2 - 4 2 0 2 0 1 - 6

This new volume, number 123, of Methods in Cell Biology looks at methods for quantitative imaging in cell biology. It covers both theoretical and practical aspects of using opt… Read more

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This new volume, number 123, of Methods in Cell Biology looks at methods for quantitative imaging in cell biology. It covers both theoretical and practical aspects of using optical fluorescence microscopy and image analysis techniques for quantitative applications.

The introductory chapters cover fundamental concepts and techniques important for obtaining accurate and precise quantitative data from imaging systems. These chapters address how choice of microscope, fluorophores, and digital detector impact the quality of quantitative data, and include step-by-step protocols for capturing and analyzing quantitative images. Common quantitative applications, including co-localization, ratiometric imaging, and counting molecules, are covered in detail. Practical chapters cover topics critical to getting the most out of your imaging system, from microscope maintenance to creating standardized samples for measuring resolution. Later chapters cover recent advances in quantitative imaging techniques, including super-resolution and light sheet microscopy. With cutting-edge material, this comprehensive collection is intended to guide researchers for years to come.

Series Editors
Preface
Chapter 1. Concepts in quantitative fluorescence microscopy
- Abstract
- 1.1 Accurate and Precise Quantitation
- 1.2 Signal, Background, and Noise
- 1.3 Optical Resolution: The Point Spread Function
- 1.4 Choice of Imaging Modality
- 1.5 Sampling: Spatial and Temporal
- 1.6 Postacquisition Corrections
- 1.7 Making Compromises
- 1.8 Communicating Your Results
- Acknowledgment
- References
Chapter 2. Practical considerations of objective lenses for application in cell biology
- Abstract
- Introduction
- 2.1 Optical Aberrations
- 2.2 Types of Objective Lenses
- 2.3 Objective Lens Nomenclature
- 2.4 Optical Transmission and Image Intensity
- 2.5 Coverslips, Immersion Media, and Induced Aberration
- 2.6 Considerations for Specialized Techniques
- 2.7 Care and Cleaning of Optics
- Conclusions
- References
Chapter 3. Assessing camera performance for quantitative microscopy
- Abstract
- 3.1 Introduction to Digital Cameras for Quantitative Fluorescence Microscopy
- 3.2 Camera Parameters
- 3.3 Testing Camera Performance: The Photon Transfer Curve
- References
Chapter 4. A Practical guide to microscope care and maintenance
- Abstract
- Introduction
- 4.1 Cleaning
- 4.2 Maintenance and Testing
- 4.3 Considerations for New System Installation
- Acknowledgments
- References
Chapter 5. Fluorescence live cell imaging
- Abstract
- 5.1 Fluorescence Microscopy Basics
- 5.2 The Live Cell Imaging Microscope
- 5.3 Microscope Environmental Control
- 5.4 Fluorescent Proteins
- 5.5 Other Fluorescent Probes
- Conclusion
- Acknowledgments
- References
Chapter 6. Fluorescent proteins for quantitative microscopy: Important properties and practical evaluation
- Abstract
- 6.1 Optical and Physical Properties Important for Quantitative Imaging
- 6.2 Physical Basis for Fluorescent Protein Properties
- 6.3 The Complexities of Photostability
- 6.4 Evaluation of Fluorescent Protein Performance in Vivo
- Conclusion
- References
Chapter 7. Quantitative confocal microscopy: Beyond a pretty picture
- Abstract
- 7.1 The Classic Confocal: Blocking Out the Blur
- 7.2 You Call that Quantitative?
- 7.3 Interaction and Dynamics
- 7.4 Controls: Who Needs Them?
- 7.5 Protocols
- Conclusions
- References
Chapter 8. Assessing and benchmarking multiphoton microscopes for biologists
- Abstract
- Introduction: Practical Quantitative 2P Benchmarking
- 8.1 Part I: Benchmarking Inputs
- 8.2 Part II: Benchmarking Outputs
- 8.3 Troubleshooting/Optimizing
- 8.4 A Recipe for Purchasing Decisions
- Conclusion
- Acknowledgments
- References
Chapter 9. Spinning-disk confocal microscopy: present technology and future trends
- Abstract
- 9.1 Principle of Operation
- 9.2 Strengths and Weaknesses
- 9.3 Improvements in Light Sources
- 9.4 Improvements in Illumination
- 9.5 Improvements in Optical Sectioning and FOV
- 9.6 New Detectors
- 9.7 A Look into the Future
- References
Chapter 10. Quantitative deconvolution microscopy
- Abstract
- Introduction
- 10.1 The Point-spread Function
- 10.2 Deconvolution Microscopy
- 10.3 Results
- Conclusion
- References
Chapter 11. Light sheet microscopy
- Abstract
- Introduction
- 11.1 Principle of Light Sheet Microscopy
- 11.2 Implementations of Light Sheet Microscopy
- 11.3 Mounting a Specimen for Light Sheet Microscopy
- 11.4 Acquiring Data
- 11.5 Handling of Light Sheet Microscopy Data
- References
Chapter 12. DNA curtains: Novel tools for imaging protein–nucleic acid interactions at the single-molecule level
- Abstract
- Introduction
- 12.1 Overview of TIRFM
- 12.2 Flow Cell Assembly
- 12.3 Importance of the Lipid Bilayer
- 12.4 Barriers to Lipid Diffusion
- 12.5 Different Types of DNA Curtains
- 12.6 Using DNA Curtains to Visualize Protein–DNA Interactions
- 12.7 Future Perspectives
- Acknowledgments
- References
Chapter 13. Nanoscale cellular imaging with scanning angle interference microscopy
- Abstract
- Introduction
- 13.1 Experimental Methods and Instrumentation
- 13.2 Image Analysis and Reconstruction
- Conclusion
- Acknowledgments
- References
Chapter 14. Localization microscopy in yeast
- Abstract
- Introduction
- 14.1 Preparing the Yeast Strain
- 14.2 Considerations for the Choice of a Labeling Strategy
- 14.3 Preparing the Sample
- 14.4 Image Acquisition
- 14.5 Results
- Summary
- Acknowledgments
- References
Chapter 15. Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM
- Abstract
- Introduction
- 15.1 Principles
- 15.2 Methods
- 15.3 Future Directions
- Acknowledgments
- References
Chapter 16. Seeing more with structured illumination microscopy
- Abstract
- Introduction
- 16.1 Theory of Structured Illumination
- 16.2 3D SIM
- 16.3 SIM Imaging Examples
- 16.4 Practical Considerations and Potential Pitfalls
- 16.5 Discussion
- References
Chapter 17. Structured illumination superresolution imaging of the cytoskeleton
- Abstract
- Introduction
- 17.1 Instrumentation for SIM Imaging
- 17.2 Sample Preparation
- 17.3 Minimizing Spherical Aberration
- 17.4 Multichannel SIM
- 17.5 Live Imaging with SIM
- Acknowledgments
- References
Chapter 18. Analysis of focal adhesion turnover: A quantitative live-cell imaging example
- Abstract
- Introduction to Focal Adhesion Dynamics
- 18.1 FA Turnover Analysis
- Acknowledgments
- References
Chapter 19. Determining absolute protein numbers by quantitative fluorescence microscopy
- Abstract
- Introduction
- 19.1 Methods for Counting Molecules
- 19.2 Protocol for Counting Molecules by Ratiometric Comparison of Fluorescence Intensity
- Conclusions
- References
Chapter 20. High-Resolution Traction Force Microscopy
- Abstract
- Introduction
- 20.1 Materials
- 20.2 Methods
- References
Chapter 21. Experimenters' guide to colocalization studies: Finding a way through indicators and quantifiers, in practice
- Abstract
- Introduction
- 21.1 An Overview of Colocalization Approaches
- Conclusion
- References
Chapter 22. User-friendly tools for quantifying the dynamics of cellular morphology and intracellular protein clusters
- Abstract
- Introduction
- 22.1 Automated Classification of Cell Motion Types
- 22.2 GUI for Morphodynamics Classification and Ready Representation of Changes in Cell Behavior Over Time
- 22.3 Results of Morphodynamics Classification
- 22.4 Geometry-based Segmentation of Cells in Clusters
- 22.5 GUI for Cell Segmentation and Quantification of Protein Clusters
- 22.6 Results for Quantifying Protein Clusters
- 22.7 Discussion
- Acknowledgments
- References
Chapter 23. Ratiometric Imaging of pH Probes
- Abstract
- Introduction
- 23.1 Currently Used Ratiometric pH Probes
- 23.2 Applications
- 23.3 Protocols
- Acknowledgments
- References
Chapter 24. Toward quantitative fluorescence microscopy with DNA origami nanorulers
- Abstract
- Introduction
- 24.1 The Principle of DNA Origami
- 24.2 Functionalizing DNA Origami Structures
- 24.3 DNA Origami as Fluorescence Microscopy Nanorulers
- 24.4 Brightness References Based on DNA Origami
- 24.5 Applications of DNA Origami Nanorulers for Visualizing Resolution
- 24.6 How to Choose an Appropriate Nanoruler for a Given Application
- References
Chapter 25. Imaging and physically probing kinetochores in live dividing cells
- Abstract
- Introduction
- 25.1 Spindle Compression to Image and Perturb Kinetochores
- 25.2 Imaging Kinetochore Dynamics at Subpixel Resolution Via Two-Color Reporter Probes
- Conclusion and Outlook
- Acknowledgments
- References
Chapter 26. Adaptive fluorescence microscopy by online feedback image analysis
- Abstract
- Introduction
- 26.1 Requirements for Adaptive Feedback Microscopy
- 26.2 Selected Applications
- Acknowledgments
- References
Chapter 27. Open-source solutions for SPIMage processing
- Abstract
- 27.1 Prerequisites
- 27.2 Overview of the SPIM Image-Processing Pipeline
- 27.3 Bead-Based Registration
- 27.4 Multiview Fusion
- 27.5 Processing on a High-Performance Cluster
- 27.6 Future Applications
- References
Chapter 28. Second-harmonic generation imaging of cancer
- Abstract
- Introduction
- 28.1 SHG Physical and Chemical Background
- 28.2 SHG Instrumentation
- 28.3 Collagen Structure as a Biomarker
- 28.4 SHG in Cancer Research
- 28.5 Quantitative Analysis of SHG Images
- Conclusion
- References
Index
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Quantitative Imaging in Cell Biology

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Jennifer Waters

Torsten Wittmann