In Vivo Fluorescence Imaging: Methods and Protocols (Methods in Molecular Biology, 1444)

✍ Scribed by Mingfeng Bai (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 216
• Category: Library

✦ Synopsis

This detailed volume includes a rich variety of applications using various instrumentations, probes, disease models, and targets in order to account for the multidisciplinary nature of the use of in vivo fluorescence imagine. The book also includes chapters on the emerging fields of cell tracking, image-guided treatment, and fluorescence imaging in the second NIR window, as well as protocols for evaluation methods before and after in vivo imaging. Written for the highly successful Methods in Molecular Biology series, chapters include brief introductions to their respective topics, lists of the necessary materials and reagents, step-by-step readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, In Vivo Fluorescence Imaging: Methods and Protocols serves as a valuable reference for researchers from numerous fields who wish to become morefamiliar with in vivo fluorescence imaging techniques.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Before In Vivo Imaging: Evaluation of Fluorescent Probes Using Fluorescence Microscopy, Multiplate Reader, and Cytotoxicity Assays
1 Introduction
2 Materials
2.1 Cell Culture Related Equipment and Reagents
2.2 Fluorescence Microscopy Equipment and Reagents
2.3 Plate Reader Equipment and Reagents
2.4 Cytotoxicity Assay Equipment and Reagents
3 Methods
3.1 Evaluation of Fluorescent Probes Using Fluorescence Microscopy
3.1.1 Preparation for Live-Cell imaging
3.1.2 Preparation for Fixed-Cell Imaging
3.1.3 Fluorescence Imaging
3.2 Evaluation of Fluorescent Probes Using Multiplate Reader
3.2.1 Quantification of Cell Uptake and Binding Specificity Using Multiplate Reader
3.2.2 Saturation Binding Assay Using Multiplate Reader
3.3 Evaluation of Fluorescent Probes Using Cytotoxicity Assays
4 Notes
References
Chapter 2: Bioconjugation Methods for Coupling Targeting Ligands with Fluorescent Dyes
1 Introduction
2 Materials
2.1 Direct Amide Coupling with Phosphonium/Uronium/Guanidinium Salt
2.2 Direct Amide Coupling with Carbodiimide
2.3 Indirect Amide Coupling Through NHS Ester
2.4 Indirect Amide Coupling Through Acyl Chloride
2.5 Thiourea Coupling with Isothiocyanate
2.6 Michael Addition with Maleimide and Thiol
2.7 Huisgen Cycloaddition (Click Chemistry) with Azide and Alkyne
2.8 Palladium-
2.9 Catalyst Free Inverse Electron Demand Diels–Alder [4 + 2] Cycloaddition
3 Methods
3.1 Direct Amide Coupling with Phosphonium/Uronium/Guanidinium Salt
3.2 Direct Amide Coupling with Carbodiimide
3.3 Indirect Amide Coupling Through NHS Ester
3.4 Indirect Amide Coupling through Acyl Chloride
3.5 Thiourea Coupling with Isothiocyanate
3.6 Michael Addition with Maleimide and Thiol
3.7 Huisgen Cycloaddition (Click Chemistry) with Azide and Alkyne
3.8 Palladium-
3.9 Catalyst Free Inverse Electron Demand Diels–Alder [4 + 2] Cycloaddition
4 Notes
References
Chapter 3: In Vivo Fluorescence Reflectance Imaging with Subcutaneous Mouse Tumor Models
1 Introduction
2 Materials
2.1 Bioconjugation of NIR Probe c(RGDyK)-ICG- Der-02
2.2 Instruments for In Vivo Imaging
2.3 Tumor Mouse Models
3 Methods
4 Notes
References
Chapter 4: FRET Imaging of Enzymatic Activities Using Smart Probes
1 Introduction
2 Materials
2.1 Peptide Synthesis
2.2 Probe Synthesis
2.3 Enzymatic Assay
2.4 In Vivo Assay
3 Methods
3.1 Peptide Synthesis
3.2 Probe Synthesis
3.3 Enzymatic Assay
3.4 In Vivo Assay
4 Notes
References
Chapter 5: Fluorescence Imaging of Inflammation in Live Animals
1 Introduction
2 Materials
2.1 Instrumentation
2.2 Fluorescence Imaging Agent
2.2.1 Inflammation Imaging Contrast Agent Synthesis General Strategy
2.2.2 Conjugation of LS601 to a 40 kDa PEG (PEG40k) (See Note 1)
2.2.3 Characterization of the LS601-­PEG40k Compound
2.2.4 Reduction of the Dyes to ROS Sensors
2.2.5 Probe Evaluation
2.3 Animal Models
2.3.1 Animals
2.3.2 Surgical Procedure to Induce Ischemia
3 In Vivo Imaging
4 Control Studies and Imaging Evaluation
4.1 Control Study
4.2 Ex Vivo Study Sample Preparation
4.3 Histology Analysis
5 Imaging Process
6 Notes
References
Chapter 6: Fluorescence Lifetime Imaging of Cancer In Vivo
1 Introduction
2 Materials
3 Methods
3.1 Animal Handling
3.2 Performing the Scan
3.3 Data Analysis
3.3.1 Fluorescence Intensity Analysis
3.3.2 Fluorescence Lifetime Analysis
3.3.3 Interpreting Fluorescence Lifetime
4 Notes
References
Chapter 7: Noninvasive Imaging of Fluorescent Reporters in Small Rodent Models Using Fluorescence Molecular Tomography
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 8: In Vivo Tumor Angiogenesis Imaging Using Peptide-Based Near-Infrared Fluorescent Probes
1 Introduction
2 Materials
3 Methods
3.1 Preparation of DBCO-­Conjugated NGR2 Peptide
3.2 Preparation of Cy5.5-NGR2 Peptide
3.3 Absorption and Emission Spectra of Cy5.5-NGR2 Peptide
3.4 Binding Specificity of Cy5.5-NGR2 to CD13 Receptor
3.5 In Vivo NIRF Optical Imaging
4 Notes
References
Chapter 9: Intraoperative Fluorescence Imaging and Multimodal Surgical Navigation Using Goggle System
1 Introduction
2 Materials
2.1 Integrated Imaging Goggle
2.2 Preoperative MRI/CT
2.3 Fluorescence Tracers
2.4 Animal and Human Requirements
3 Methods
3.1 Preclinical SLN Surgical Guidance
3.2 Preclinical Image-Guided Tumor Resection
3.3 Goggle-Aided Intraoperative Imaging in Human Patients
3.4 Fluorescence-
3.5 Intraoperative Fluorescence Imaging Integrated with Preoperative Tomography-Based Surgical Navigation
3.6 Conclusions
4 Notes
References
Chapter 10: Utilization of Near-Infrared Fluorescent Imaging for Pharmaceutically Relevant Applications
1 Introduction
2 Materials
2.1 Materials Used for NIR Imaging of Nanoemulsions
2.2 Materials for Biologic Material Quantification and Release Studies in Hydrogels Using NIR Imaging
2.3 Materials Needed for NIR Assessment of Cellular Uptake of Fluorescent Nanoemulsions
2.4 Materials Needed for In Vivo and Ex Vivo Assessment of Nanoemulsions in a Mouse Inflammation Model
3 Methods
3.1 Methods for Imaging Nanoemulsions
3.1.1 Standard Calibration Curve
3.1.2 Sample Preparation for Nanoemulsions
3.1.3 Method for Imaging of NIR Dye-­Loaded Nanoemulsions
3.2 Methods for Biologic Material Quantification and Release Studies in Hydrogels Using NIR Imaging
3.2.1 Standard NIR Imaging Calibration Curve for Bacteria
3.2.2 Sample Preparation for Bacteria Quantification
3.2.3 Sample Preparation for Bacteria Release Studies
3.2.4 Quantification by NIR Imaging of Bacteria Content and Release
3.3 Methods for Assessment of Cellular Uptake of Fluorescent Nanoemulsions by NIR Imaging
3.4 Methods for In Vivo and Ex Vivo Assessment of Nanoemulsions in a Mouse Inflammation Model by NIR Imaging
3.4.1 Animal Treatment and Preparation for Imaging
3.4.2 Image Analysis for Quantification of Signal in Animals
4 Notes
References
Chapter 11: In Vivo Cell Tracking Using Two-Photon Microscopy
1 Introduction
2 Materials
2.1 Animals
2.2 Reagents
2.3 Equipment
3 Methods
3.1 Two-Photon Microscopy of Whole-Mount Tissues Containing Cells Labeled with Vital Fluorescent Dyes
3.2 Two-Photon Microscopy of Whole-Mount Tissues Containing Cells Genetically Marked by Fluorescent Proteins
3.3 In Vivo Tracking of Label-Free Resident Cells in Murine Tissues via Third Harmonic Generation Microscopy (THG)
4 Notes
References
Chapter 12: Small-Animal Imaging Using Diffuse Fluorescence Tomography
1 Introduction
2 Materials
2.1 MRI-DFT Imaging system
2.2 Reagents
2.3 Software
3 Methods
3.1 Animal Preparation
3.2 MRI-DFT Imaging Protocol (48 h After Injection as Per Above)
3.2.1 Positioning and System Calibration
3.2.2 Imaging
3.3 MRI and Optical Data Processing
3.3.1 MRI Image Processing
3.3.2 Optical Data Processing
3.4 Combining Data to Reconstruct MRI-DFT Images
3.4.1 Data Calibration
3.4.2 Reconstruct Images
3.4.3 Visualization and Image Processing
3.5 Advanced MRI-DFT Imaging Techniques
4 Notes
References
Chapter 13: In Vivo Metal Ion Imaging Using Fluorescent Sensors
1 Introduction
2 Materials
2.1 CS790AM Purity Analysis
2.2 CS790AM Aliquoting
2.3 Animals and Animal Preparation
2.4 Injection
2.5 Imaging
2.6 Blood Collection, Perfusion, and Organ Harvesting
2.7 Blood Analysis
3 Methods
3.1 CS70AM Quality Control (See Note 1)
3.1.1 TLC Purity Analysis
3.1.2 1H-NMR Purity Analysis
3.2 CS790AM Aliquots (See Note 3)
3.3 Fur Removal (If Necessary)
3.4 Preparation of Injectables
3.5 Intraperitoneal Injection Procedure
3.6 Imaging Procedure and Image Analysis
3.7 Blood Collection, Perfusion, Organ Harvesting, and Organ Imaging
3.8 Blood Analysis
4 Notes
References
Chapter 14: Using Fluorescence Imaging to Track Drug Delivery and Guide Treatment Planning In Vivo
1 Introduction
2 Materials
2.1 Nanoparticle Preparation and Characterization
2.2 Four-Step Imaging to Track Drug Delivery
2.2.1 Establishing Orthotopic Prostate Cancer Model
2.2.2 Validation of Orthotopic Prostate Cancer Model and Quantification of Tumor Volume
2.2.3 In Vivo Whole-Body Fluorescence Imaging (2D Imaging)
2.2.4 CT-FMT Co-registration (3D Tomography Imaging)
2.2.5 In Situ and Ex Vivo Fluorescence Imaging
2.2.6 Confocal Imaging of Frozen Tumor Tissue Slice
2.3 CT-FMT Co-registration Image-Guided Treatment Planning
2.4 In Vivo Therapeutic Efficacy Study
2.4.1 Polymerase Chain Reaction Assay
2.4.2 Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling Assay
3 Methods
3.1 Nanoparticle Preparation and Characterization
3.2 Four-Step Imaging to Track Drug Delivery
3.2.1 Establishing Orthotopic Prostate Cancer Model
3.2.2 Measurement of Tumor Volume Using MRI
3.2.3 In Vivo Whole-Body Fluorescence Imaging (2D Imaging)
3.2.4 CT-FMT Imaging and Image Co-registration
3.2.5 In Situ and Ex Vivo Fluorescence Imaging
3.2.6 Confocal Imaging of Frozen Tumor Tissue Slice
3.3 Using CT-FMT Co-registration Image to Guide Treatment Planning
3.3.1 FMT Quantification
3.3.2 Assessment of Multiple-Dosing siRNA Delivery
3.4 In Vivo Therapeutic Efficacy Study
3.4.1 Polymerase Chain Reaction Assay
3.4.2 Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling Assay
4 Notes
References
Chapter 15: In Vivo Fluorescence Imaging in the Second Near-Infrared Window Using Carbon Nanotubes
1 Introduction
2 Materials
2.1 Preparation of Biocompatible SWCNT Solution
2.2 In Vivo SWCNT Administration
2.3 In Vivo NIR-II Imaging System
3 Methods
3.1 Preparation of Biocompatible SWCNT Solution for Injection
3.2 In Vivo NIR-II Fluorescence Imaging Setup
3.3 In Vivo Administration of SWCNT Solution and Image Acquisition
3.4 Post-acquisition Processing of In Vivo NIR-II Fluorescence Images
4 Notes
References
Chapter 16: Ex Vivo Imaging, Biodistribution, and Histological Study in Addition to In Vivo Imaging
1 Introduction
2 Materials
2.1 Ex Vivo Imaging and Biodistribution Study
2.2 Histological Study
3 Methods
3.1 Ex Vivo Imaging
3.2 Biodistribution Study
3.3 Histological Study
3.3.1 Embed Tissue into Frozen Blocks
3.3.2 Preparation of Frozen Sections
3.3.3 Staining and Fluorescence Imaging of Frozen Sections
4 Notes
References
Chapter 17: Experimental Design and Data Analysis of In Vivo Fluorescence Imaging Studies
1 Introduction
2 Experimental Design
2.1 Three Rs
2.2 Hypothesis Formulation
2.3 Sources of Variation
2.4 Basic Principles
2.5 Design Types
2.5.1 Completely Randomized Design
2.5.2 Randomized Complete Block Design
2.5.3 Factorial Design
2.5.4 Repeated Measures Design
2.6 Sample Size and Power
2.6.1 Effect Size
2.6.2 Standard Deviation
2.6.3 Significance Level
2.6.4 Power
Example 1: Independent Sample Experiment
Example 2: Paired Sample Experiment
Example 3: Repeated Measures Experiment
3 Data Analysis
3.1 One-Tailed and Two-Tailed Tests
3.2 T Tests
3.3 ANOVA
3.4 ANCOVA
3.5 Repeated Measures ANOVA or ANCOVA
3.6 Chi-Square Test
3.7 Summary Statistics and Inference Statistics
3.7.1 Standard Deviation vs. Standard Error
3.7.2 P-Value
3.7.3 Confidence Interval
3.8 Multiplicity Adjustment
Example 1: Continuous Data Without Repeated Measures
Example 2: Continuous Data with Repeated Measures
Example 3: Binary Data (Without Repeated Measures)
4 Conclusion
References
Index