Multiplexed Imaging: Methods and Protocols (Methods in Molecular Biology, 2350)

✍ Scribed by Eli Zamir (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 347
• Category: Library

✦ Synopsis

This volume provides a collection of state-of-the-art approaches addressing key aspects of multiplexed imaging. Chapters focus on labeling and imaging techniques for multiplexed imaging, as well as on the application of these techniques for the study of cells and tissues. Written in the highly successful Methods in Molecular Biology series format, chapters include 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, Multiplexed Imaging: Methods and Protocols aims to be helpful for researchers interested in implementing multiplexed imaging or in developing novel, cutting-edge multiplexed imaging approaches.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Strategies for Multiplexed Biosensor Imaging to Study Intracellular Signaling Networks
1 Introduction
2 Overview of Biosensor Design
2.1 Modulating FRET between a Pair of FPs
2.2 Modulating Dimerization-Dependent Fluorescent Protein (ddFP) Proximity
2.3 Modulating the Fluorescence of a Single FP
2.4 Inducing FP Translocation
3 Multiplexing Strategies
3.1 Imaging Multiple FRET-Based Biosensors
3.1.1 Orthogonal FRET Pairs
3.1.2 Fluorescence-Lifetime Imaging Microscopy (FLIM)
3.1.3 FRET Between Identical Fluorophores (Homo-FRET)
3.2 Imaging Multiple Single-Fluorophore Biosensors
3.3 Maximizing the Possibilities by Combining Designs and Approaches
4 Conclusion and Perspectives
References
Chapter 2: Six-Color Confocal Immunofluorescence Microscopy with 4-Laser Lines
1 Introduction
2 Materials
2.1 Preparation of Tissue Samples
2.2 Staining of Tissue Samples
2.2.1 Reagents
2.3 Embedding of Tissue Samples
2.4 Microscopy
3 Methods
3.1 Preparation of Tissue Samples
3.2 Staining of Tissue Samples
3.2.1 Preparation of the Tissue Slides
3.2.2 Streptavidin/Biotin and Fc Block
3.2.3 Staining of the Tissue
3.3 Embedding of Tissue Samples
3.4 Microscopy
4 Notes
References
Chapter 3: Multiplexed Imaging of Posttranslational Modifications of Endogenous Proteins in Live Cells
1 Introduction
2 Materials
2.1 Plasmids
2.2 Cell Lines
2.3 Transfection Reagents
2.4 Cell Culture and Inhibitors
2.5 Microscopy and Image Analysis
3 Methods
3.1 Establishing Cell Lines Stably Expressing Mintbodies
3.2 Microscopic Observation to Analyze the Effect of a Suv420H1/2 Inhibitor on Histone H4 Lysine 20 Methylation States
3.3 Time-Course Analysis for Evaluating the Effect of a Histone Deacetylase Inhibitor
3.4 Mintbodies Fused with a Fluorescent Protein and SNAP-Tag for Multiplexed Imaging
4 Notes
References
Chapter 4: Multiplex Imaging of Rho GTPase Activities in Living Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Transfection, Retrovirus Production, and Transduction
2.3 Imaging Experiments
3 Methods
3.1 Production of Double-Stable and Inducible NIR-Rac1 FRET Biosensor Cell Lines
3.1.1 Production of Retrovirus
3.1.2 Infection and Selection of Cells
3.2 Multiplex Imaging of GTPase Activities Using Cyan-Yellow and NIR FRET Biosensors
3.2.1 Coverslip Preparation for Imaging
3.2.2 Induction of Biosensor Expression
3.2.3 Imaging of Biosensors in Living Cells
3.2.4 Processing of Multiplex Image Data to Determine GTPase Activity
4 Notes
References
Chapter 5: Multicolor Localization-Based Super Resolution Microscopy
1 Introduction
2 Materials
2.1 OxEA Imaging Buffer
2.2 Imaging Chambers
2.3 Ultra Clean Coverslips
2.4 Antibodies
2.5 Microscope
2.6 ThunderStorm
3 Methods
3.1 Sample Preparation
3.1.1 Staining of the Cells
3.1.2 Staining of the Tissue Sections
3.1.3 Buffer Preparation
3.2 Measurement
3.3 Calibration for the Correction of Chromatic Aberration
3.4 Post-Processing
4 Notes
References
Chapter 6: Multiplexed Tissue Tomography
1 Introduction
2 Materials
2.1 Tissue Collection
2.1.1 Whole Tumor Excision
2.1.2 Core Needle Biopsy Collection
2.2 Fixation
2.3 Tissue Embedding
2.3.1 Agarose Gel Embedding for Whole Tumors
2.3.2 Agarose Cassette Preparation for a Core Needle Biopsy
2.4 Macrosectioning
2.5 Antibody-Dye Conjugation
2.6 Antibody Staining
2.7 Optical Clearing
2.8 Imaging
3 Methods
3.1 Antibody-Dye Conjugation
3.2 Tissue Collection
3.2.1 Whole Tumor Excision
3.2.2 Core Needle Biopsy Collection
3.2.3 Fixation
3.3 Tissue Embedding
3.3.1 Agarose Gel Embedding and Macrosectioning for Whole Tumors
3.3.2 Agarose Cassette Preparation for a Core Needle Biopsy
3.4 Antibody Staining
3.5 Optical Clearing
3.6 Imaging
4 Notes
References
Chapter 7: Multicolor 3D Confocal Imaging of Thick Tissue Sections
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
3 Methods
3.1 Preparation of Reagents
3.1.1 Fixative
3.1.2 TBS-T
3.1.3 Permeabilization, Blocking, Washing, and Staining Buffers
3.1.4 Mounting Media
3.2 Femur Dissection and Processing
3.3 Vibratome Sectioning of Femurs
3.4 Immunostaining of Femur Sections
3.5 Mounting Femur Sections
4 Notes
References
Chapter 8: Multiphoton Deep-Tissue Imaging of Micrometastases and Disseminated Cancer Cells Using Conjugates of Quantum Dots a...
1 Introduction
2 Materials
2.1 Components for Nanoprobe Fabrication
2.2 Components for Quality Control and Functional Tests
2.2.1 Cell Lines and Animal Models
2.2.2 Protein Extraction
2.2.3 Immunohistochemistry
2.3 Special Equipment
3 Methods
3.1 Nanoprobe Fabrication (Fig. 1)
3.1.1 QDs Water Solubilization
3.1.2 QD´s Functionalization and Purification
3.1.3 QD´s Quality Control
3.1.4 Reduction of the Intermolecular Disulfide Bonds Within Single-Domain Antibody Dimers
3.1.5 Conjugation of sdAb-Cys with PEG-Modified Colloidal QDs
Conjugation of sdAb-SH with Amino-Modified QDs Using the Sulfo-SMCC Reaction
Conjugation of sdAb-SHs with Hydroxyl-Modified QDs Using the PMPI Reaction
3.2 Preparation of Relevant Experimental Models
3.2.1 Tumor Animal Models
3.2.2 Immunohistochemistry in Thin Sections
3.2.3 Immunohistochemistry in Thick Sections
3.3 Confocal and Two-Photon Microscopies
3.4 Quality Control of sdAb-HER2-QD (Fig. 2)
3.5 Quality Control of sdAb-CEA-QD (Fig. 3)
3.6 Deep Tissue Imaging of Solid Tumors (Fig. 4)
3.7 Disseminated Tumor Cell Imaging (Fig. 5)
3.8 Micrometastase Imaging (Fig. 6)
4 Notes
References
Chapter 9: Multiplexed Imaging for Immune Profiling on Human FFPE Material
1 Introduction
2 Materials
2.1 Slide Preparation, Deparaffinization, and Rehydration of the Samples
2.2 Antigen Retrieval
2.3 Primary Antibodies and Antibody Dilution
2.4 General and Additional Reagents
2.5 Akoya Manual System and Primary Antibody Labeling
2.6 Slide Mounting
2.7 Imaging
2.8 Digital Image Analysis
3 Methods
3.1 Pre-work Procedures
3.2 Slide Preparation, Deparaffinization, and Rehydration of the Samples
3.3 Antigen Labeling
3.3.1 First Antibody: CD1a
3.3.2 Second Antibody: CD208
3.3.3 Third Antibody: CD15
3.3.4 Fourth Antibody: CD123
3.3.5 Fifth Antibody: CD68
3.3.6 Sixth Antibody: Pan-Cytokeratin/E-Cadherin Cocktail
3.3.7 DAPI Staining
3.4 Spectral Library
3.5 Creating Scanning Protocol
3.6 Whole Slide Imaging
3.7 Field/MSI Imaging
3.8 Image Analysis Project Establishment
3.9 Batch Analysis and Image Control
3.10 Data Structure and Data Analysis
4 Notes
References
Chapter 10: Method for Multiplexed Dynamic Intravital Multiphoton Imaging
1 Introduction
2 Materials
2.1 Choosing the Optimal Set of Fluorophores
2.2 Imaging and Anesthesia
2.3 Two-Photon Laser-Scanning Microscope Setup
2.4 Data Analysis
3 Methods
3.1 Acquiring the Fingerprints of the Fluorophores and Signals Combination
3.2 Microscope Setup Preparation
3.3 Multiplexed Dynamic Intravital Imaging
3.4 Spectrally Unmixing the Imaging Data
4 Notes
References
Chapter 11: Fourier Multiplexed Fluorescence Lifetime Imaging
1 Introduction
1.1 Background
1.2 FmFLIM Method
1.3 Lifetime Analysis of FmFLIM Image Data
1.4 FmFLIM Tomography
2 Materials
2.1 Fluorescence Lifetime Standard Slides
2.2 Tomographic Imaging Sample Mounting
2.3 Multiwavelength Excitation Laser Source
2.4 Detection System
2.5 Signal Processing Circuits (All Components Need to Have 50 Omega Impedance)
2.6 Michelson Interferometer Components
2.7 Confocal Microscope Components (See Note 7)
2.8 Tomography Imaging Components (See Note 8)
2.9 General Optical Components
2.10 System Control
3 Methods
3.1 Set up the Multiwavelength Excitation Laser Beam
3.2 Set Up the Interferometer
3.3 Build the FmFLIM Confocal Microscope (Fig. 3)
3.4 Imaging Control, Signal Processing, and Data Acquisition
3.5 Calibrate Lifetime Channels with Fluorescence Standards
3.6 Analyze Lifetime Images
3.7 Tomographic FmFLIM Imaging
4 Notes
References
Chapter 12: Bimolecular Fluorescence Complementation (BiFC) and Multiplexed Imaging of Protein-Protein Interactions in Human L...
1 Introduction
2 Materials
2.1 BiFC Vector Designing and Cloning Constructs
2.2 Cell Culture and Plasmid Transfection
2.3 Confocal Imaging
2.4 Data Analysis
3 Methods
3.1 Transfection of BiFC Plasmids
3.2 Detect Fluorescent Signals
3.3 Semiquantitative BiFC Analyses
3.4 Data Interpretation
4 Notes
References
Chapter 13: Out-of-Phase Imaging after Optical Modulation (OPIOM) for Multiplexed Fluorescence Imaging Under Adverse Optical C...
1 Introduction
1.1 Fighting Against Ambient Light
1.1.1 Spectral Analysis
1.1.2 Dynamic Excitation
1.2 Fighting Against Autofluorescence
1.2.1 The Growth Medium
1.2.2 The Optical Filter Set
1.2.3 The Wavelength Range
1.2.4 Chemical Treatments
1.2.5 Photobleaching
1.3 Multiplexed Fluorescence Imaging
1.3.1 Spectral Discrimination
1.3.2 Temporal Discrimination
2 Materials
2.1 Modulatable Light Sources
2.2 Home-Built Setup for OPIOM Implementation in Epifluorescence Microscopy
2.3 Home-Built Setup for OPIOM Implementation in Single Plane Illumination Microscopy (SPIM)
2.4 Home-Built Setup for Speed-OPIOM Implementation in Epifluorescence Microscopy
2.5 Home-Built Setup for Speed-OPIOM Implementation in Macroscale Fluorescence Imaging
2.6 Home-Built Setup for Speed-OPIOM Implementation in Fluorescence Endomicroscopy
3 Methods
3.1 Measurement of Light Intensities
3.2 Video Acquisition
3.3 Extraction of the (Speed-)OPIOM Images
3.4 Conclusion
4 Notes
4.1 OPIOM Implementation in Epifluorescence Microscopy
4.2 OPIOM Implementation in Single Plane Illumination Microscopy (SPIM)
4.3 Speed-OPIOM Implementation in Epifluorescence Microscopy
4.4 Speed-OPIOM Implementation in Macroscale Fluorescence Imaging
4.5 Speed-OPIOM Implementation in Fluorescence Endomicroscopy
4.6 Absolute Measurement of Light Intensities
4.7 Calibration of Light Intensities by Actinometry
References
Chapter 14: Multicolor Bioluminescence Imaging of Subcellular Structures and Multicolor Calcium Imaging in Single Living Cells
1 Introduction
2 Materials
2.1 Imaging of Subcellular Structures
2.2 Calcium Imaging in Single Living Cells
2.3 Equipment
3 Methods
3.1 Multicolor Bioluminescence Imaging of Subcellular Structures in Single Living Cells
3.1.1 Microscope Setup
3.1.2 Selection of Objective Lens
3.1.3 Selection of Emission Filters
3.1.4 Checking the Expression Levels of eNLs in Cells
3.1.5 Taking Bioluminescence Images of eNLs and Nluc
3.2 Multicolor Calcium Imaging in Single Living Cells
3.2.1 Checking Expression Levels of eNL(Ca2+)S in Cells
3.2.2 Taking Bioluminescence Images of eNL(Ca2+)S
4 Notes
References
Chapter 15: Nanoparticles for In Vivo Lifetime Multiplexed Imaging
1 Introduction
2 Materials
2.1 Nanoparticles
2.2 Imaging System
3 Methods
3.1 Nanoparticles
3.2 Imaging
3.3 Computational Analysis
4 Notes
References
Chapter 16: Versatile On-Demand Fluorescent Labeling of Fusion Proteins Using Fluorescence-Activating and Absorption-Shifting ...
1 Introduction
2 Materials
2.1 General
2.2 Protein Labeling in Live and Fixed Cells
2.3 Expression and Purification of Recombinant FAST Protein
2.4 Protein Labeling in Zebrafish Embryo
3 Methods
3.1 Rapid and Reversible Labeling of FAST-Tagged Proteins in Live Mammalian Cells for Fluorescence Microscopy Experiments
3.2 Rapid Labeling of FAST-Tagged Proteins in Live Cells for Flow Cytometry Experiments
3.3 Labeling of FAST-Tagged Proteins in Fixed Mammalian Cells for Fluorescence Microscopy Experiments
3.4 Quantification of FAST-Tagged Proteins in Solutions
3.5 Expression and Purification of FAST Protein
3.6 Labeling FAST-Tagged Proteins in Live Zebrafish Embryo
4 Notes
References
Chapter 17: UltraPlex Hapten-Based Multiplexed Fluorescent Immunohistochemistry
1 Introduction
2 Materials
2.1 UltraPlex Fluorescent Immunohistochemistry
2.2 UltraPlex Slide Imaging
3 Methods
3.1 UltraPlex Fluorescent Immunohistochemistry Manual Staining Procedure
3.2 UltraPlex Fluorescent Immunohistochemistry-Automated Staining Procedure
3.3 Preparation of UltraTag Calibration Microspheres
3.4 UltraPlex Slide Imaging Procedure
3.5 UltraPlex Digital Image Overlay Procedure
4 Notes
References
Chapter 18: Multimodal Approach for Cancer Cell Investigation
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Immunofluorescence
2.3 Video Microscopy
2.4 Atomic Force Microscopy
3 Methods
3.1 Cell Culture
3.2 Immunofluorescence
3.3 Video Microscopy
3.4 Atomic Force Microscopy
4 Notes
References
Chapter 19: Multiplexed Fourier Transform Infrared and Raman Imaging
1 Introduction
2 Materials
2.1 Preparation of Cells for Multiplex Analysis
2.2 Preparation of Tissue for Multiplex Analysis
3 Methods
3.1 Preparation of Cells for Multiplex Analysis
3.2 Preparation of Tissue for Multiplex Analysis
3.3 Identification of the Region of Interest
3.4 Infrared Imaging
3.5 Raman Imaging
3.6 Image Registration
3.7 Basic Data Analysis
3.7.1 Principal Component Analysis (PCA)
3.7.2 Statistical Heterospectroscopy (SHY)
4 Notes
References
Chapter 20: Multiplexed Imaging Mass Spectrometry of Histological Staining, N-Glycan and Extracellular Matrix from One Tissue ...
1 Introduction
2 Materials
2.1 Dewaxing Solutions
2.2 Hematoxylin and Eosin Staining Solutions (See Note 1)
2.3 MALDI-IMS Solutions
2.4 Tissue Clearing Solutions
2.5 Enzyme Solutions
2.6 Instrumentation and Tools
3 Methods
3.1 Tissue Heating and Dewaxing
3.2 Hematoxylin and Eosin Staining
3.3 Antigen Retrieval for PNGase F Application
3.4 Slide Scanning
3.5 PNGase F Application by the M3 TM-Sprayer
3.6 PNGase F Incubation for on-Tissue Digestion
3.7 MALDI Matrix Application by the TM-Sprayer
3.8 Tissue Clearing of Matrix between Enzymes
3.9 Antigen Retrieval for Collagenase Type III Application
3.10 Collagenase Type III Application by the M3 TM-Sprayer
3.11 Collagenase Type III Incubation for on-Tissue Digestion
3.12 MALDI Matrix Application by the TM-Sprayer
4 Notes
References
Chapter 21: Multiplexed Raman Imaging in Tissues and Living Organisms
1 Introduction
2 Materials
2.1 SERS NP Synthesis
2.2 Functionalization and Ligand Conjugation
2.3 Conjugating Targeting Ligands
2.4 In Vitro Targeting Validation
2.5 In Vivo SERS NP Multiplexing
2.6 Data Analysis
3 Methods
3.1 SERS NP Synthesis
3.2 Thiol Functionalization
3.3 Conjugating Targeting Ligands
3.4 In Vitro Targeting Validation
3.5 In Vivo SERS NP Multiplexing
3.6 Data Analysis
4 Notes
References
Correction to: Out-of-Phase Imaging after Optical Modulation (OPIOM) for Multiplexed Fluorescence Imaging Under Adverse Optica
Index