Imaging from Cells to Animals In Vivo

Cover
Half Title
Series Page
Title Page
Copyright Page
Table of Contents
Preface
The Editors
List of Contributors
Section I Overview of Imaging Methods and Instrumentation
Chapter 1 Fluorescence Microscopy Techniques
1.1 Introduction
1.2 Principles of Fluorescence Imaging
1.2.1 Photophysics of Fluorescence
1.2.2 Effects of the Host Environment on Fluorescent Molecules
1.2.3 Effects of Fluorescent Molecules on Host Environment
1.2.4 Fluorophore Types
1.2.5 Endogenous Fluorophores (EnF)
1.2.6 Exogenous Fluorophores (ExF)
1.2.6.1 Fluorescent Dyes
1.2.6.2 Quantum Dots
1.2.6.3 Fluorescence Indicators
1.3 Principles of Microscopy
1.3.1 Image Formation and Magnification
1.3.2 Numerical Aperture and Spatial Resolution
1.3.3 Fluorescence Microscopy Techniques for In Vivo Studies
1.3.3.1 Deconvolution
1.3.3.2 Confocal Microscopy
1.3.3.3 Light Sheet Microscopy
1.3.3.4 Multiphoton Microscopy
1.4 Summary
References
Chapter 2 Intravital Microscopy
2.1 Instrumentation
2.1.1 Optically Transparent vs. Opaque Specimens
2.1.2 Spatial Resolution
2.1.3 Temporal Resolution
2.1.4 Depth
2.2 Surgical Preparation
2.2.1 Digestive System
2.2.2 Respiratory and Cardiovascular System
2.2.3 Reproductive System
2.2.4 Urinary System
2.2.5 Nervous System
2.2.6 Immune System
2.2.7 Other Tissues
2.3 Motion Artifact Control
2.4 Anesthesia Control
2.5 Temperature Control
2.6 Fluorescent Labeling
2.6.1 Antibodies and Lectins
2.6.2 Organic Dyes
2.6.3 Inorganic Dyes: Quantum Dots
2.6.4 Fluorescent Protein
2.7 Pitfalls and Shortcomings
2.8 Toward High Resolution: IVM Correlative Light and Electron Microscopy (CLEM)
2.9 Future Developments
References
Chapter 3 An Introduction to Live-Cell Super-Resolution Imaging
3.1 The Diffraction Limit
3.2 Super-Resolution Microscopy Techniques
3.2.1 Structured Illumination Microscopy
3.2.1.1 Axial Resolution
3.2.1.2 Temporal Resolution
3.2.1.3 Hardware and Sample Preparation
3.2.1.4 Examples of Live-Cell SIM
3.2.1.5 Limitations of SIM
3.2.1.6 Other Microscopy Implementations Based on the SIM Principle
3.2.2 Stimulated Emission Depletion Microscopy
3.2.2.1 Axial Resolution
3.2.2.2 Temporal Resolution
3.2.2.3 Hardware and Sample Preparation
3.2.2.4 Case Studies of Live-Cell STED Microscopy
3.2.2.5 Limitations of STED Microscopy
3.2.2.6 Other Microscopy Techniques Based on the Principles of STED
3.2.3 Single Molecule Localization Microscopy
3.2.3.1 Axial resolution
3.2.3.2 Temporal Resolution
3.2.3.3 Hardware and Sample Preparation
3.2.3.4 Case Studies of Live-Cell SMLM
3.2.3.5 Limitations of SMLM
3.2.3.6 Promising Approaches for SMLM
3.3 Emerging Techniques for Live-Cell Super-Resolution Microscopy
3.3.1 Hardware Developments
3.3.2 Analytical developments
3.3.3 Labeling Developments
3.4 Super-Resolution Data Evaluation
3.5 Conclusion
References
Chapter 4 Endoscopic Optical Coherence Tomography: Technologies and Applications
4.1 Introduction
4.2 Design of OCT Endoscope
4.2.1 Micro-Optics
4.2.2 Scanning Methods
4.2.2.1 Side-View Scanning
4.2.2.2 Forward-View Scanning
4.2.3 Miniaturized OCT Endoscopes
4.2.4 OCT Endoscopes for Imaging Large Lumens
4.2.4.1 Balloon OCT Endoscope
4.2.4.2 Capsule
4.3 From Small Animal Study to Clinical Application
4.3.1 Small Animal Study
4.3.2 OCT Imaging in Airways
4.3.2.1 Obstructive Lung Disease
4.3.2.2 Pulmonary Fibrosis
4.3.2.3 Lung Cancer
4.3.3 OCT Imaging in the Gastrointestinal Tract
4.3.3.1 Esophagus
4.3.4 OCT Imaging in the Cardiovascular System
4.3.5 Other Applications
References
Chapter 5 Bioluminescence
5.1 Introduction
5.2 The Chemistry of Bioluminescence
5.3 A Diversity of Luciferases for Bioluminescent Imaging
5.4 Applications of Bioluminescence for In Vivo Imaging
5.4.1 Traditional In Vivo Bioluminescent Imaging Using Firefly Luciferase and d-Luciferin
5.4.2 In Vivo Bioluminescent Imaging Without Substrate
5.4.3 In Vivo Bioluminescent Imaging at Longer Wavelengths
5.4.4 Multiplexed In Vivo Bioluminescent Imaging
5.4.5 In Vivo Bioluminescent Imaging Using a Caged Luciferin
5.4.6 Split Luciferases for In Vivo Bioluminescent Imaging
5.4.7 Bioluminescence Resonance Energy Transfer (BRET)
5.4.8 Fluorescence by Unbound Excitation from Luminescence (FUEL)
5.4.9 Emerging Animal Models That Permit Endogenous In Vivo Bioluminescent Imaging
5.5 Instrumentation for Bioluminescent Imaging
5.6 The Advantages and Disadvantages of Bioluminescent Imaging
5.7 Conclusion and the Future of In Vivo Bioluminescent Imaging
References
Chapter 6 Macroscopic Fluorescence Imaging
6.1 Introduction
6.2 Experimental Techniques
6.3 Theoretical Methods
6.3.1 The Forward Problem
6.3.1.1 The General TD Forward Problem
6.3.1.2 Frequency Domain
6.3.1.3 The Asymptotic Limit
6.3.1.4 Spatial Frequency Domain
6.3.2 The Inverse Problem
6.3.2.1 Direct Time Domain Inversion
6.3.2.2 Asymptotic Time Domain Inversion
6.3.2.3 Optimal Estimator
6.3.2.4 Spectral vs. Lifetime Multiplexing
6.4 Computational Methods
6.5 Biological Applications
6.5.1 Intrinsic Fluorescence
6.5.2 Extrinsic Fluorescence
6.5.3 Activatable Fluorescence
6.6 Summary
References
Chapter 7 Optical Coherence Tomography
7.1 Introduction
7.2 Principles of OCT
7.2.1 Low-Coherence Interferometry
7.2.2 OCT System Variants
7.2.2.1 Time-Domain Optical Coherence Tomography
7.2.2.2 Frequency-Domain Optical Coherence Tomography
7.2.3 OCT System Performance
7.2.3.1 Axial Resolution
7.2.3.2 Transverse Resolution
7.2.3.3 System Sensitivity
7.2.3.4 Phase Stability
7.2.3.5 Acquisition Rates
7.3 Contrast Techniques
7.3.1 Refractive Index
7.3.2 Polarization-Sensitive OCT
7.3.3 Spectroscopic OCT
7.3.4 Optical Coherence Elastography
7.3.5 Optical Coherence Tomography Angiography
7.3.5.1 Phase-Signal Based OCTA Techniques
7.3.5.2 Intensity-Signal Based OCTA Techniques
7.3.5.3 Complex-Signal Based OCTA Techniques
7.3.6 Exogenous Contrast Agents for OCT
7.3.7 Multimodal OCT
7.4 OCT Applications: From Cellular to Animal Imaging
7.4.1 Cellular Imaging
7.4.2 Tissue Engineering
7.4.2.1 Imaging of Scaffolds
7.4.2.2 Cell Dynamics
7.4.3 Small Animal Imaging
7.4.3.1 Ophthalmology
7.4.3.2 Cardiology
7.4.3.3 Musculoskeletal Imaging
7.4.3.4 Dermatology
7.5 Conclusion
7.6 Acknowledgments
References
Chapter 8 Multiscale Photoacoustic Imaging
8.1 Introduction
8.2 Spatial Scalability of PAI
8.3 Photoacoustic Imaging at the Organelle Level
8.4 Photoacoustic Imaging at the Cellular Level
8.5 Photoacoustic Imaging at the Tissue Level
8.6 Photoacoustic Imaging at the Organ Level
8.7 Photoacoustic Imaging of the Small-Animal Whole Body
8.8 Frontiers in Photoacoustic Imaging
8.9 Conclusion
Acknowledgments
References
Chapter 9 Fluorescence Lifetime: Techniques, Analysis, and Applications in the Life Sciences
9.1 Introduction
9.1.1 Fluorescence
9.1.2 Theory
9.2 Instrumentation
9.2.1 Fluorometry and FLIM
9.2.2 Spectral and Polarization Resolved FLIM
9.2.3 Laser Scanning Microscopes and FLIM
9.2.4 Camera-Based FLIM Microscopes
9.2.5 Challenges in FLIM Instrumentation for Live-Animal Imaging
9.2.6 Key Points
9.3 Analysis
9.3.1 Exponential Decay Curve Fitting
9.3.2 Fitting Routines
9.3.2.1 Maximum Likelihood Estimation (MLE) – Nonlinear Least-Square (NLS) Fitting
9.3.2.2 Rapid Lifetime Determination – Levenberg Marquardt Algorithm
9.3.2.3 Global Analysis
9.3.2.4 Moment Analysis
9.3.2.5 Stretched Exponentials
9.3.2.6 Laguerre Expansion: Model Free Deconvolution
9.3.2.7 Blind Deconvolution
9.3.2.8 Maximum Entropy Method
9.3.2.9 Bayesian Analysis
9.3.3 Phasor Analysis
9.3.4 Software for FLIM Curve Fitting
9.3.4.1 Commercial Options
9.3.4.2 FLIMFit
9.3.4.3 SLIM Curve Library
9.3.5 Standards
9.3.6 Key points
9.4 Applications
9.4.1 FRET
9.4.2 Autofluorescence and Clinical Applications
9.4.2.1 Development of FLIM signatures
9.4.2.2 FLIM Endoscopy
9.4.3 Fluorescence Lifetime Correlation Spectroscopy (FLCS)
9.4.4 Flow Cytometry (FCM) Aided with Fluorescence Lifetime
9.4.5 Superresolution
9.4.6 Voltage Sensitive Dyes
9.4.7 Environment: Viscosity, Refractive Index, Temperature, pH, pO2, and ROS
9.4.8 Key points
9.5 Discussion
9.6 Conclusion
9.7 Glossary
Acknowledgments
References
Section II Imaging Cellular Behavior
Chapter 10 Imaging Cell Metabolism: Analyzing Cellular Metabolism by NAD(P)H and FAD
10.1 Introduction
10.2 Multiphoton Fluorescence Lifetime Imaging Microscopy (MP-FLIM), Time-Correlated Single Photon Counting (TCSPC)
10.3 Optimizing Autofluorescent NAD(P)H and FAD FLIM Acquisition
10.3.1 Excitation Wavelengths, Filters, Laser Power vs. Acquisition Times
10.3.2 Testing Possibility of Photodamage
10.3.3 Pretesting Fixed Cells vs. Live
10.3.4 Exploring the Application of “Stitching” and “Z-Stacks”
10.3.5 The Choice of Imaging Same FOVs for Controls and Treatment
10.4 FLIM-Fitting Process in B&H SPCImage Software
10.4.1 Instrument Response Function, Automatic and/or Measured?
10.4.2 Fitting Models and Parameters
10.4.3 Phasor Plots
10.4.4 Data Export Choices
10.5 Processing SPCImage: Exported Data in ImageJ/Fiji Software
10.5.1 Preparing Photon Images for ROI Selection
10.5.2 Selecting Three Sets of ROIs: Mitochondrial, Cytosolic, and Segmented Cells
10.5.3 An ImageJ/Fiji Macro Extracts Data by ROI/Cell to Generate “Results”
10.6 NAD(P)H and FAD Biological Background
10.7 FLIM-Based Redox Ratio (FLIRR) Assay
10.8 Quantitative Analysis, Charting, and Statistics
10.8.1 Separate Analysis of Whole-Cell, Mitochondrial, and Cytosolic Data
10.8.2 Segmented Cell Data and Categorization Based on FLIRR Response to Treatment
10.9 Application of FLIRR Assay to a Tumor Xenograft Mouse Model
10.10 Summary
Acknowledgment
References
Chapter 11 Intravital Imaging of Cancer Cell Migration In Vivo
11.1 Introduction
11.2 Traditional Tools for Studying Cancer Progression and Dissemination Are Limited
11.3 Intravital Imaging
11.4 Surgical Engineering Expands the Capabilities of Intravital Imaging
11.5 Large-Volume High-Resolution Intravital Imaging
11.6 Types of Motility: Collective, Single Cell, and Single Cell Streaming
11.7 Single Cell Streaming Motility Results in Hematogenous Dissemination
11.8 Collective Vascular Invasion
11.9 Surgical Engineering Enables Direct Visualization of the Lung Vasculature, Serially
11.10 Conclusion
Acknowledgments
References
Chapter 12 Imaging Cellular Signaling In Vivo Using Fluorescent Protein Biosensors
12.1 Introduction
12.2 Fluorescent Protein Biosensors
12.3 Challenges of IVM of Biosensors – Imaging Living Tissues
12.3.1 The Limitations of Multiphoton Microscopy
12.3.2 Depth-Dependent Signal Attenuation
12.3.3 Tissue Motion
12.3.4 Physiological Maintenance of Small Animals
12.4 Challenges of IVM of Biosensors – In Vivo Expression of FP Biosensors
12.4.1 Transgenic FP Biosensor Mice
12.4.2 In Vivo Gene Transfer via Viral Transduction
12.4.3 Transplantation of Ex-Vivo Transduced Tissue
12.5 Caveats and Considerations for IVM of FRET Biosensors
12.5.1 Ratiometric Measurements of FRET In Vivo
12.5.2 Validating In Vivo FRET Measurements
12.5.3 Measuring FRET In Vivo
12.6 IVM Future Directions
Acknowledgments
References
Chapter 13 Imaging Cell Adhesion and Migration
13.1 Introduction
13.1.1 Cell Motility Cycle
13.1.2 FRET Biosensors for Imaging Signaling Pathway Activation in Real Time
13.1.2.1 RhoGTPase Biosensors
13.2 Imaging Cell Motility
13.2.1 Reconstituting Cell Motility Behavior In Vitro
13.2.2 Imaging Migration In Vivo
13.2.3 In Vivo Imaging and FRET Biosensors
13.3 Imaging Cell Adhesion
13.3.1 Imaging Cellular Adhesion In Vitro
13.3.1.1 Super-Resolution Microscopy
13.3.2 Adhesion In Vivo
13.4 Conclusions and Perspectives
13.5 Acknowledgments
References
Chapter 14 Imaging the Living Eye
14.1 Ocular Anatomy
14.2 Fundus Camera
14.3 Confocal Scanning Laser Ophthalmoscope
14.4 Optical Coherence Tomography
14.4.1 OCT Angiography
14.4.2 Visible-Light OCT
14.4.2.1 Retinal Oximetry
14.4.2.2 Doppler vis-OCT
14.4.2.3 Calculating the Inner Retinal Metabolic Rate of Oxygen
14.4.2.4 Validation of Metabolic Imaging Using vis-OCT
14.4.2.5 Animal Studies with vis-OCT
14.5 Photoacoustic Ophthalmoscopy
14.6 Adaptive Optics
14.7 Summary
References
Section III Whole-Organ and Whole-Organism Imaging
Chapter 15 Heart Imaging
15.1 Echocardiography of Mammalian Hearts
15.1.1 Echocardiographic Modes
15.1.2 Echocardiographic Measurements
15.2 Optical Mapping of Mammalian Hearts
15.2.1 Langendorff Heart
15.2.2 Voltage Sensitive Dyes and Calcium Indicators
15.2.3 Optical Mapping
15.3 Optical Manipulation of Heart Electrical Activity
15.3.1 Optogenetics in Cardiovascular Research
15.3.2 Optogenetic Resynchronization and Defibrillation
15.3.3 All Optical Platform to Probe and Control Heart Activity
15.4 Nonlinear Imaging
15.4.1 Two-Photon Fluorescence Microscopy
15.4.2 Functional Imaging at Subcellular Level
15.4.3 Intravital Imaging of Cardiac Function at the Single-Cell Level
15.5 Whole Heart Imaging in Zebrafish Larva
15.5.1 Single Plane Illumination Microscopy
15.5.2 High-Resolution Reconstruction of the Beating Zebrafish Heart
References
Chapter 16 Visualizing Hepatic Immunity through the Eyes of Intravital Microscopy
16.1 Introduction
16.2 Hepatic Morphology
16.2.1 Structure and Organization
16.2.1.1 Ex Vivo × In Vivo Images
16.2.2 Cells In Vivo: Location and Distribution
16.3 Immunostaining for In Vivo Imaging
16.3.1 Common Cell Markers under Homeostasis
16.3.1.1 Hepatocytes Can Be Identified by Their Autofluorescence
16.3.1.2 Liver Vessels Labeling Strategy
16.3.1.3 Liver Immune Cells Can Be Labeled by Their Surface Antigen Expression
16.4 Liver Imaging Acquisition
16.4.1 The Procedure
16.4.2 Experimental Design and Probes Administration
References
Chapter 17 Optical Imaging of the Mammalian Oviduct In Vivo
17.1 Introduction
17.1.1 Why We Need to Image the Oviduct
17.1.2 In Vitro and Ex Vivo Imaging Approaches
17.1.3 The Demand for In Vivo Imaging
17.1.4 An Overview of This Chapter
17.2 Fluorescence Microscopy
17.2.1 Fluorescent Labeling and Imaging Setup
17.2.2 In Vivo Sperm Imaging in the Isthmus
17.3 Optical Coherence Tomography
17.3.1 OCT and Its Imaging Contrast
17.3.2 In Vivo Imaging Setup
17.3.3 Structural Imaging of Tissues and Cells
17.3.4 Functional Assessment of the Muscle Contraction
17.3.5 Functional Imaging of the Ciliary Dynamics
17.4 Discussions
17.4.1 Current Challenges and Technical Improvements
17.4.2 Future Applications
17.5 Conclusions
Acknowledgments
References
Chapter 18 Immune System Imaging
18.1 Introduction
18.2 Imaging Bone Marrow
18.2.1 The Hematopoietic Stem Cell Niche
18.2.2 Neutrophil and Monocyte and Mobilization
18.2.3 Lymphocyte Dynamics in the Bone Marrow
18.2.4 Thrombopoiesis
18.3 Imaging the Thymus
18.3.1 Introduction
18.3.2 Overview of Thymocyte Development
18.3.3 Imaging the Thymic Microenvironment via 2PM
18.3.4 Thymocyte Motility During Positive and Negative Selection
18.3.5 Chemokine Control of Thymocyte Movement
18.3.6 Imaging the Thymus – Summary
18.4 Imaging the Lymph Node
18.4.1 Lymph Node Structure
18.4.2 Lymphocyte Interactions in High Endothelial Venules
18.4.3 Dynamics of T Cells, B Cells, and Dendritic Cells in the Lymph Node
18.4.4 B Cells and Humoral Responses
18.4.5 Immune Regulation in the Lymph Node
18.4.6 Imaging the Lymphatic Vasculature
18.4.7 Lymph Node and Lymphatic Imaging – Summary
18.5 Imaging the Spleen
18.5.1 Introduction
18.5.2 Spleen Structure
18.5.3 Imaging Lymphocytes in the Spleen
18.5.4 Imaging B Cell Behavior in the Spleen
18.5.5 Visualizing Responses to Pathogens in the Spleen
18.6 Concluding Remarks
References
Chapter 19 Imaging Living Organisms
19.1 Background
19.1.1 Technical Developments
19.2 In Vivo 2PM Imaging
19.2.1 Vital Points in Animal Surgeries
19.2.2 Imaging Procedures
19.3 Optical Aberrations in Brain Tissue
19.3.1 Deep Imaging
19.3.2 Light Refraction and Aberrations
19.4 Future Directions
References
Chapter 20 Live Imaging of Zebrafish
20.1 Introduction – Why and When to Use Zebrafish for In Vivo Imaging
20.2 Approaches for Labeling Zebrafish
20.2.1 Ubiquitous Labeling
20.2.2 Labeling a Defined Population of Cells
20.2.3 Mosaic Labeling
20.2.4 Clonal and Single-Cell Labeling
20.2.5 Biosensors in Cells
20.3 Imaging Techniques
20.3.1 Basic Principles in Light-Based Live Imaging Methods
20.3.2 Wide-Field Light Microscopy
20.3.3 Confocal and Multiphoton Laser Scanning Microscopy
20.3.4 Light Sheet Fluorescence Microscopy
20.4 Applications
20.4.1 Cell Behavior and Fate During Morphogenesis
20.4.2 Cell Biology
20.4.3 Brain Activity and Behavior
20.4.4 High Content Screening
20.5 Data Analysis
20.6 Future Directions
References
Chapter 21 Whole-Body Fluorescence Imaging in Cancer Research
21.1 Introduction
21.2 In Vivo Imaging of Biodistribution of Novel Anticancer Agents
21.3 Assessment of Photobleaching of Photosensitizer during PDT
21.4 In Vivo Monitoring of Tumor Growth and Response to Therapy Using Fluorescent Proteins
21.5 Functional Fluorescence Imaging of Tumors In Vivo
21.6 Conclusions
Acknowledgments
References
Chapter 22 Large-Scale Fluorescence Imaging in Neuroscience
22.1 Introduction
22.2 Multiphoton Microscopy
22.2.1 Physical Principle
22.2.2 Realization
22.2.3 Recent Advances in Multiphoton Microscopy
22.2.3.1 Imaging Depth
22.2.3.2 Imaging Speed
22.2.3.3 Multicolor Imaging
22.3 Temporal Focusing – Light-Sculpting an Arbitrary PSF
22.4 Applications of Light-Sculpting in In Vivo Neuronal Activity Imaging
22.4.1 Whole-Brain Imaging of C. elegans
22.4.2 Large-Volume Cortical Imaging in Awake Mice
22.5 Conclusion and Outlook
References
Index