Confocal Microscopy: Methods and Protocols (Methods in Molecular Biology, 2304)

✍ Scribed by Joseph Brzostowski (editor), Haewon Sohn (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 363
• Category: Library

✦ Synopsis

This volume provides a wide range of imaging protocols that can be tailored to specific organisms or cell-types. Chapters guide readers through fixed-cell, live-cell, phenotype screening, super-resolution, intravital imaging techniques, and fluorescence life-time imaging microscopy (FLIM). 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 cutting-edge, Confocal Microscopy: Methods and Protocols aims to ensure successful results in the further study of this vital field.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: Advances in Confocal Microscopy and Selected Applications
1 Introduction
2 Resolution of Intensity
2.1 Sample Preparation
2.1.1 Labeling with Stains
Penetrance
Specificity
Bias
Accuracy
2.1.2 Labeling with Genetically Encodable Tags
Penetrance
Specificity
Bias
Accuracy
2.1.3 Labeling Structures vs Specific Biomolecules
2.2 Detector Efficiency
3 Temporal Resolution
3.1 Resonant Scanners
3.2 Slit Confocal
3.3 Swept-Field Confocal
3.4 Spinning Disk Confocal
4 Spatial Resolution
4.1 Deconvolution
4.1.1 Mechanism of Basic Deconvolution
4.1.2 Weaknesses of Basic Deconvolution
4.1.3 Workflow of Basic Deconvolution
4.1.4 A Note on Conflicting Deconvolution Literature
4.2 Superresolution Imaging
4.2.1 Airyscan Imaging
4.2.2 Depletion Technologies
5 Chromatic Resolution
5.1 Bandpass Filters, Notch Filters, and Dichroic Mirrors
5.2 Adjustable Chromatic Filtering Systems
5.3 Multispectral Imaging
6 Resolution in Additional Orthogonal Dimensions
6.1 Fluorescence Lifetime Imaging
7 Resolution of Analysis Pipelines
8 Resolution of Dynamic Properties of Ensemble Systems
8.1 Photobleaching Recovery Experiments
8.2 Other Intensity Fluctuation Tools
9 Conclusions
References
Chapter 2: Choosing Fluorescent Probes and Labeling Systems
1 Introduction
1.1 Fluorescence Basics
1.2 Labeling Systems
2 Materials
2.1 Labeling Proteins, Peptides, and Thiolated Biomolecules
2.2 Immunofluorescence
2.3 Labeling HaloTag Expressing Mammalian Cells
2.4 Transfecting Mammalian Cells with Fluorescence Protein Expression Vectors
2.5 Extrinsic Labeling of IFP2.0 Expressing Mammalian Cells with Biliverdin
3 Methods
3.1 Labeling Proteins, Peptides, and Thiolated Biomolecules (See Note 2)
3.2 Immunofluorescence
3.3 Labeling HaloTag Expressing Mammalian Cells (See Note 14)
3.4 Transfecting Mammalian Cells with Fluorescence Protein Expression Vectors (See Note 15)
3.5 Extrinsic Labeling of IFP2.0 Expressing Mammalian Cells with Biliverdin
4 Notes
References
Chapter 3: General Considerations for Acquiring a Three-Color Image by Laser Scanning Confocal Microscopy
1 Introduction
2 Materials
3 Methods
3.1 Set Parameters for Three Color Imaging on Three Tracks
3.2 Acquire a Three-Dimensional Image
3.3 Setting the Focus of the Condenser
4 Notes
References
Chapter 4: Microfabricated Devices for Confocal Microscopy on Biological Samples
1 Introduction
1.1 Microfabricated Structures and Biology
1.2 Microfabrication Techniques
1.3 Design Considerations for Confocal Microscopy
1.4 Application Examples
2 Materials
2.1 Template Fabrication
2.2 PDMS Molding
2.3 Reproducing Molds
2.4 In Situ Well Formation
2.5 Device Assembly
3 Methods
3.1 Template Fabrication
3.2 PDMS Molding
3.3 Reproducing Molds
3.4 Stamping Directly onto a Coverglass
3.5 Device Assembly-PDMS Channels on a Glass Coverslip
4 Notes
References
Chapter 5: ZEISS Airyscan: Optimizing Usage for Fast, Gentle, Super-Resolution Imaging
1 Introduction
2 Materials
3 Methods
3.1 Airyscan Imaging
3.1.1 Turn On the System and Launch the Zen (Black) Software
3.1.2 Turn on Lasers
3.1.3 Place the Sample on the Stage and Locate Your Sample
3.1.4 Set the Airyscan Light Path Configuration for Image Acquisition
3.1.5 Aligning the Airyscan Detector
3.1.6 Set Parameters in Channels Window for Acquisition
3.1.7 Saving Images
3.1.8 Tuning off the Microscope
3.1.9 Airyscan Image Processing
3.2 Airyscan FAST Imaging
3.2.1 Set the Airyscan FAST Light Path Configuration
3.2.2 Aligning the Airyscan Detector for the FAST Mode
3.2.3 Acquiring Airyscan FAST Images
4 Notes
References
Chapter 6: High-Resolution Multicolor Imaging of Mitochondria in Lymphocytes
1 Introduction
2 Materials
2.1 Isolation of Primary Cells
2.2 Cell Staining
2.3 Coverslip Preparation and Mounting
2.4 Microscopy, Image Deconvolution and Analysis
3 Methods
3.1 Cell Isolation
3.2 Cell Staining and Adhesion to Coverslip
3.2.1 Cell Staining in Solution: LIVE/DEAD Fixable Green Dead Cell Stain and MitoTracker Red CMXRos
3.2.2 Adhesion of Cells to Coverslip
3.2.3 Cell Staining on Coverslip: TOM20-ATTO-647N and DAPI
3.3 Mounting Coverslip to Slide
3.4 Microscopy, Image Deconvolution, and Analysis
3.4.1 Microscopy Instrumentation and Imaging
3.4.2 Image Deconvolution
3.4.3 Image Analysis
4 Notes
References
Chapter 7: Protein-Retention Expansion Microscopy (ExM): Scalable and Convenient Super-Resolution Microscopy
1 Introduction
2 Materials
2.1 Fixation and Staining
2.2 AcX Anchoring
2.3 Gel Embedding
2.4 Digestion
2.5 Expansion and Imaging
3 Methods
3.1 Fixation and Staining
3.2 AcX Anchoring
3.3 Gel Embedding
3.3.1 Gel Embedding for Cultured Cells
3.3.2 Gel Embedding for Brain Tissue Slices
3.4 Digestion
3.5 Expansion and Imaging
4 Notes
References
Chapter 8: Analysis of B Cell Receptor-Mediated Antigen Extraction by B Lymphocytes from Plasma Membrane Sheets Using Confocal...
1 Introduction
2 Materials
2.1 Preparing Antigen-Containing PMS Using Miniwells
2.2 Internalization of PMS-Bound Antigen by B Cells
2.3 Image Acquisition Using Confocal Microscope
2.4 Image Processing and Analysis
3 Methods
3.1 Preparing Antigen-Containing PMS Using Miniwells
3.2 Internalization of PMS-Bound Antigen by B Cells
3.3 Image Acquisition Using Confocal Microscope
3.4 Image Processing and Analysis
4 Notes
References
Chapter 9: Analysis of Intracellular Vesicles in B Lymphocytes: Antigen Traffic in the Spotlight
1 Introduction
2 Materials
2.1 Cells
2.2 Fixed Samples
2.3 Live Samples
2.4 Transfections
2.5 Microscope
2.6 Software for Image Analysis
3 Methods
3.1 Visualising Antigen Traffic in Fixed Cells
3.1.1 Preparation of Immunofluorescence Samples
3.1.2 Imaging of the Immunofluorescence Samples
3.2 Analyzing Antigen Traffic in Live Cells
3.2.1 Transfections
3.2.2 Preparing the Samples
3.2.3 Imaging of Live Samples
3.3 Image Analysis
3.3.1 Deconvolution by Batch Processing
3.3.2 Analysis of Clustering
3.3.3 Colocalization Analysis in Fiji
3.3.4 Spot Colocalization and Tracking in Imaris
4 Notes
References
Chapter 10: Visualizing Key Signaling Components of Macropinocytosis and Phagocytosis Using Confocal Microscopy in the Model O...
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Preparation for Microscopy
2.3 Confocal Microscopy
2.4 Yeast Preparation
3 Methods
3.1 Cell Culture
3.2 Labeling Yeast with Alexa 633
3.3 Monitoring Temporospatial Distribution of Key Signaling Components During Macropinocytosis and Phagocytosis with Standard ...
3.3.1 Seed Cells into a 4-Well Chamber for Imaging
3.3.2 Monitoring Temporospatial Distribution of Key Signaling Components During Macropinocytosis and Phagocytosis with Standar...
3.3.3 Monitoring Signaling Events of Phagocytosis Using the AiryScan Technique
4 Notes
References
Chapter 11: Imaging GPCR-Mediated Signal Events Leading to Chemotaxis and Phagocytosis
1 Introduction
2 Materials
2.1 Cell Culture and Chemical Stocks
2.2 Time-Lapse Confocal Imaging
2.3 EZ-TAXIScan System
3 Methods
3.1 Time-Lapse Imaging for LPS Chemotactic Signaling
3.2 Micropipette Assay for Folate Chemotaxis
3.3 EZ-TAXIScan Assay for Folate Chemotaxis
3.4 Engulfment Assay of Live Bacteria Particles by Confocal Imaging
4 Notes
References
Chapter 12: High-Throughput Imaging of Arrays of Fluorescently Tagged Yeast Mutant Strains
1 Introduction
2 Materials
2.1 Yeast Mutant Arrays and Query Strain
2.2 General Stock Solutions
2.3 Solid Media for SGA Replica Pinning Protocol
2.4 Liquid Media for HTP Sample Preparation and Imaging
2.5 Accessories and Equipment
2.5.1 SGA
2.5.2 HTP Imaging
3 Methods
3.1 SGA Strategy for Introducing Fluorescent Markers into Yeast Mutant Strain Arrays
3.2 High-Throughput Preparation of Yeast Cells and HTP Imaging
3.2.1 Day 0: Preparing Plasticware and Media (Done Beforehand)
3.2.2 Day 1: Preparing Yeast Overnight Cultures
3.2.3 Day 2: Subculturing
3.2.4 Day 3: HTP Imaging of Plates of the Deletion Collection Array
3.2.5 Day 3: HTP Imaging of Plates of the TS Collection Array
3.3 Image Analysis
4 Notes
References
Chapter 13: Visualizing the Dynamics of T Cell-Dendritic Cell Interactions in Intact Lymph Nodes by Multiphoton Confocal Micro...
1 Introduction
2 Materials
3 Methods
3.1 DC Isolation and Peptide Loading
3.2 Naïve CD4+ T Cell Isolation and Primary Amine Labeling
3.3 Adoptive Transfer
3.4 Minimally Invasive Surgery for the Intravital Microscopy (IVM) of Popliteal Lymph Node
3.5 Acquisition for Intravital Microscopy (IVM) of Popliteal Lymph Node
3.6 Data Analysis for Intravital Microscopy (IVM) of Popliteal Lymph Node
3.6.1 Correct Thermal Drift and Improve Image Quality by Applying Deconvolution
3.6.2 Track Cells and Calculate Colocalization
3.6.3 Calculate the Colocalization and Cell-Cell Distance
4 Notes
References
Chapter 14: Studying Neuronal Biology Using Spinning Disc Confocal Microscopy
1 Introduction
2 Materials
3 Methods
3.1 Coating Plates with Matrigel
3.2 Thawing Human-Induced Pluripotent Stem Cells (i3PSCs)
3.3 Passaging i3PSCs
3.4 Cryopreservation
3.5 Generating Fluorescent i3PSCs
3.6 Differentiation of i3Neurons
3.7 Axon Morphology: Imaging and Measurement
3.7.1 Sample Preparation
Neuronal Branching
Neurite Length
3.7.2 Axon Morphology Imaging
3.7.3 Axon Morphology Analysis
4 Notes
References
Chapter 15: Method for Acute Intravital Imaging of the Large Intestine in Live Mice
1 Introduction
2 Materials
2.1 Animals
2.2 Surgical Materials
2.3 Devices and Reagents for Intravital Microscopy
2.4 Microscopes and Components (See Note 2)
3 Methods
3.1 Surgery to Image Through the Serosa
3.2 Surgery to Image from the Lumen and Intestinal Epithelium (See Note 5)
3.3 Imaging Parameters
3.4 Image Processing
4 Notes
References
Chapter 16: Fluorescence Lifetime Imaging as a Noninvasive Tool to Study Plasmodium Falciparum Metabolism
1 Introduction
2 Materials
3 Methods
3.1 Sample Preparation
3.2 Microscope Setup
3.3 Image Acquisition A-Comparison Between Uninfected and Infected RBCs
3.4 Image Acquisition B-Metabolic Inhibition of Trophozoite-Stage iRBCs
3.5 FLIM Analysis
4 Notes
References
Chapter 17: Developing Analysis Protocols for Monitoring Intracellular Oxygenation Using Fluorescence Lifetime Imaging of Myog...
1 Introduction
2 Materials
2.1 Transfection of Living Cells with Myo-mCherry
2.2 Cell Treatment with Rotenone and Antimycin A
2.3 Imaging Setup
2.4 IRF Measurements (Multiphoton Microscopy)
2.5 Controlled Experimental Environment with a Range of Stable Oxygen Concentrations for Imaging and Calibration
3 Methods
3.1 Transfection of Living Cells with Myo-mCherry
3.2 Fluorescence Two-Photon Image Acquisition
3.3 IRF Measurement (Multiphoton Microscopy)
3.4 Controlled Experimental Environment with a Range of Stable Oxygen Concentrations for Imaging
3.5 Myo-mCherry Calibration and Cell Treatment with Rotenone and Antimycin A
3.6 Measurements of the Oxygen Partial Pressure (pO2) at Each Imposed Oxygen Concentration
3.7 FLIM Analysis
3.8 Obtaining the Intracellular pO2 from Lifetime Data at Each Imposed Oxygen Concentration
3.9 Statistical Analysis
4 Notes
References
Chapter 18: FLIM Imaging for Metabolic Studies in Live Cells
1 Introduction
2 Materials
3 Methods
3.1 Expression of the Sensor Proteins in U2-OS Cells (Day 1)
3.2 Turning on the FLIM System (Day 2)
3.3 Donor-Only Control Using CP-TMR-SMX
3.4 Measure Donor Lifetime with Acceptor
3.5 Control Experiments Using the Inhibitor Sulfapyridine
4 Notes
References
Chapter 19: A Step-by-Step Guide to Instant Structured Illumination Microscopy (iSIM)
1 Introduction
2 Materials
2.1 Hardware and Image Acquisition Software
2.2 Cell Culture
2.3 Immunocytochemistry
3 Methods
3.1 Image Acquisition Protocol (Specific for MetaMorph Acquisition Software)
3.2 Image Post Processing
3.2.1 Image Deconvolution
3.2.2 Image De-striping Using Fourier Space Notch Filers
4 Notes
References
Correction to: Confocal Microscopy
Index