Correlative light and electron microscopy V /

Correlative Light and Electron Microscopy V, Volume 187 in the Methods in Cell Biology series highlights advances in the field, with this new volume presenting interesting chapters on timely topics, including Orthotopic brain tumor models derived from glioblastoma stem-like cells, RNA sequencing in...

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Bibliographic Details
Corporate Author:	ScienceDirect (Online service)
Other Authors:	Müller-Reichert, Thomas, 1962- (Editor), Verkade, Paul (Editor)
Format:	eBook
Language:	English
Published:	London : Academic Press is an imprint of Elsevier, 2024.
Series:	Methods in cell biology ; v. 187.
Subjects:	Electron microscopy. Light. Microscopie électronique. Lumière. electron microscopy. light (energy) Electronic books.
Online Access:	Connect to the full text of this electronic book

Table of Contents:

Intro
Correlative Light and Electron Microscopy V
Copyright
Contents
Contributors
Preface
Chapter One: How to apply the broad toolbox of correlative light and electron microscopy to address a specific biological ...
1. Introduction
2. Instrumentation and materials
2.1. Selection of chemically fixed cells for electron microscopy (EM)
2.1.1. Light microscopy of cells plated on Aclar
2.1.2. Fixation and thin-layer embedding for electron microscopy (EM)
2.1.3. Selection and analysis of re-mounted cells
2.2. High-pressure freezing/freeze substitution of cultured cells (cryo-immobilization approach)
2.2.1. Seeding cells plated on sapphire discs
2.2.2. High-pressure freezing of cells
2.2.3. (Automatic) freeze substitution of frozen cells
2.2.4. Selection of cells by light microscopy (LM)
2.3. On-section labeling (immuno-EM approach)
2.3.1. Sample fixation
2.3.2. Resin embedding and sectioning
2.3.3. Labeling of resin sections for CLEM and imaging
2.3.4. Gelatine/sucrose embedding for Tokuyasu cryo-sectioning
2.3.5. Labeling of cryo-sections for CLEM and imaging
2.4. Overlay of fluorescence and transmission electron microscopy images
3. Methods
3.1. Selection of chemically fixed cells for electron microscopy (broadest approach)
3.1.1. Preparation of cells plated on Aclar
3.1.2. Fixation and thin-layer embedding of cells
3.1.3. Selection of cells by light microscopy (LM)
3.2. High-pressure freezing/freeze substitution of cultured cells (cryo-immobilization approach)
3.2.1. Preparation of cells plated on sapphire discs
3.2.2. High-pressure freezing of cells
3.2.3. (Automatic) freeze substitution (AFS) of frozen cells
3.2.4. Sectioning of the sample blocks
3.2.5. Selection of cells by light microscopy (LM).
3.2.6. Imaging of cells of interest by electron microscopy (EM)
3.3. On-section immunolabeling of ultrathin sections for combined fluorescence light and electron microscopy (EM)
3.3.1. Sample fixation
3.3.2. Resin embedding and sectioning
3.3.3. Labeling of resin sections for CLEM and imaging
3.3.4. Gelatine/sucrose embedding for Tokuyasu cryo-sectioning
3.3.5. Labeling and imaging of cryo-sections
3.4. Overlay of fluorescence and transmission electron microscopy images
4. Discussion
4.1. Application of the broadest CLEM approach
4.2. Application of the cryo-immobilization approach
4.3. Application of on-section labeling (immuno-EM) approach
4.4. Overlay of images
4.5. Concluding remarks
Acknowledgments
References
Chapter Two: Some tips and tricks for a Correlative Light Electron Microscopy workflow using stable expression of fluores ...
1. Introduction
2. Instrumentation and materials
2.1. Cell culture
2.2. Light microscopy
2.3. Electron microscopy
2.4. Post processing
3. Methods
3.1. Cell culture and fixation
3.2. Laser scanning confocal microscopy
3.3. TEM sample processing
3.3.1. Epon infiltration and embedding
3.3.2. Lift-off
3.3.3. Trimming
3.3.4. Sectioning
3.3.5. EM correlation
4. Conclusion
Acknowledgments
References
Chapter Three: Targeting of membrane proteins with fluoronanogold probes for high-resolution correlative microscopy
1. Introduction
2. Instrumentations and materials
2.1. Expression of biotinylated proteins in rat organotypic hippocampal slices
2.2. FNG labeling and silver enhancement
2.3. EM sample preparation and data acquisition
3. Methods
3.1. Expression of biotinylated proteins in rat organotypic hippocampal slices
3.2. FNG labeling and silver enhancement.
3.3. EM sample preparation and data acquisition
3.4. Image analysis
4. Discussion
Acknowledgments
References
Chapter Four: Correlative light and electron microscopy at defined cell cycle stages in a controlled environment
1. Introduction
2. Instrumentation and materials
2.1. Live cell imaging for CLEM under constant gas concentration and temperature
2.2. High-pressure freezing
2.3. Freeze substitution and resin embedding
2.4. Sectioning, post-staining, and electron tomography
3. Methods
3.1. Maintaining cells in a controlled environment until high-pressure freezing
3.2. Live cell imaging in a controlled environment
3.3. High-pressure freezing
3.4. Freeze substitution and resin embedding
3.5. Pre-checking cells of interest in the resin block using light microscopy
3.6. Correlation of live cell imaging with TEM-based tomography
3.7. Identification of small and low-abundant intracellular structures using EM tomography
4. Summary and future perspectives
Acknowledgments
References
Chapter Five: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) culture and sample preparation for correlative ...
1. Introduction
2. Instrumentation and materials
2.1. Cell culture
2.2. Virus work
2.2.1. Virus culture
2.2.2. Viral stock preparation
2.2.3. Viral stock quantification
2.3. Carbon-coated coverslips for CLEM
2.4. Light microscopy
2.5. Electron microscopy
2.5.1. Staining for EM
2.5.2. EM processing
3. Methods
3.1. Cell culture
3.1.1. Virus culture and purification
3.1.2. Virus quantitation
3.2. Carbon-coated coverslips for CLEM
3.2.1. Preparation of carbon-coated coverslips for CLEM
3.2.2. Coating of coverslips
3.2.3. Seeding cells onto the prepared coverslips
3.2.4. Infection of cells seeded on coverslips.
3.3. Light microscopy
3.4. Processing for EM
4. Concluding remarks
References
Chapter Six: A correlated light and electron microscopy approach to study the subcellular localization of phosphorylated ...
1. Introduction
2. Methods
2.1. Light microscopy
2.1.1. Chemical fixation
2.1.2. Immunofluorescence
2.2. Electron microscopy
2.2.1. Electron microscopy amplification processing
2.2.2. Electron microscopy embedding
2.2.3. Ultramicrotomy and TEM
2.3. Image analysis
3. Instrumentation and materials
3.1. Light microscopy
3.1.1. Chemical fixation
3.1.2. Immunofluorescence
3.2. Electron microscopy
3.2.1. Electron microscopy amplification processing
3.2.2. Electron microscopy embedding
3.2.3. Ultramicrotomy and TEM
3.3. Image analysis
4. Results and discussion
4.1. General considerations for CLEM on specific tissues
4.2. Cryostat section preservation (LM)
4.3. Probes and accessibility of antigens
4.4. Structural preservation for labeling
4.5. Probe amplification for electron microscopy
4.6. Counterstaining of sections
4.7. Data acquisition and correlation of LM and EM images
5. Conclusion
Acknowledgments
References
Chapter Seven: Array tomography of in vivo labeled synaptic receptors
1. Introduction and rationale
2. Instrumentation and materials
2.1. C. elegans strains and maintenance
2.2. C. elegans preparation for microinjection
2.3. The microinjection setup and procedure
2.4. In vivo labeling of C. elegans via microinjection
2.5. Live labeling with cell-permeable SNAP-TMR dye
2.6. High-pressure freezing, freeze substitution, and embedding
2.7. Serial sectioning, landmark staining, SIM imaging and processing
2.8. Scanning electron microscopy
2.9. Image correlation
2.10. Image stack export and 3D reconstruction.
3. Methods
3.1. C. elegans strains and maintenance
3.2. C. elegans preparation for microinjection
3.3. The microinjection setup and procedure
3.4. In vivo labeling of C. elegans via microinjection
3.5. Live labeling with cell-permeable SNAP-TMR dye
3.6. High-pressure freezing (HPF), freeze substitution (FS), and embedding
3.7. Serial sectioning, landmark staining, SIM imaging and processing
3.8. Scanning electron microscopy
3.8.1. SEM image adjustment and montage
3.8.2. SEM image alignment
3.9. Image correlation
3.10. Image stack export and 3D reconstruction
4. Results
5. Advantages
6. Limitations
7. Optimization and troubleshooting
8. Alternative methods/procedures
Acknowledgments
References
Chapter Eight: Correlative cryo-microscopy pipelines for in situ cellular studies
1. Introduction
2. Materials and instrumentation
2.1. Cell culture
2.2. Plunge-freezing and clipping of grids
2.3. Imaging with FLM and confocal
2.4. Imaging with cryoFLM
2.5. Imaging with cryoET
2.6. Correlative softwares
3. Method
3.1. Cells on grids preparation
3.2. Plunge-freezing
3.3. Cryo-fluorescence imaging
3.4. Fluorescence imaging at RT
3.5. Correlative imaging in the cryoEM
3.6. Post-acquisition correlation
4. Troubleshooting and optimization
5. Discussion and conclusion
6. Summary
7. Safety considerations and standards
Acknowledgments
References
Chapter Nine: Building a super-resolution fluorescence cryomicroscope
1. Introduction
2. Overview
2.1. Cryo-specific design considerations
2.1.1. Laser light source
2.1.2. Samples
2.1.3. LED light source
2.1.4. Objective lens
2.1.5. Sample stage
2.2. Parts list
2.3. Software
3. Construction
3.1.1. Mounting
3.1.2. Laser illumination path
3.1.3. Imaging path.