Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells
September 7th, 2018
A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.
This method helps to answer key questions in the biology field about how bio-proteins who are in cellular membranes to create environments that favor bio-replication and assembly.The two technical cornerstones that are combined here are first, the correlation between live cell imaging and electron microscopy, and second, high pressure freezing.Although this method has been designed and successfully utilized to tackle virus source interactions, it can be easily generalized to a range of applications in cell biology.Visual demonstration of this method is critical as there are many steps that require practical experience with success, especially Cryo-immobilization by high-pressure freezing and the subsequent steps.Begin by placing patterned sapphire discs with an alphanumeric pattern etched onto one of their surfaces into a 15 milliliter conical centrifugation tube, and washing the discs thoroughly with ethanol.Place the washed sapphire discs in a Petri dish lined with filter paper for drying, and use long, slim tweezers to transfer the dried sapphire discs onto glass slides.Using an inverted microscope at 4x magnification, confirm that the pattern of coordinates on every sapphire disc is readable.Then, sterilize the discs with an ultraviolet crosslinker for five minutes.In a biosafety cabinet, add half of the cell culture medium to a cell culture dish, and use long tweezers to carefully transfer the sapphire discs into the dish.Then, seed 1x10
Overview
This article presents a correlative light electron microscopy (CLEM) method to visualize virus-induced intracellular structures. By integrating light microscopy (LM) and electron microscopy (EM), researchers can gain insights into virus-host interactions.
Key Study Components
Area of Science
- Cell Biology
- Microscopy Techniques
- Virus-Host Interactions
Background
- The method combines live cell imaging with electron microscopy.
- High pressure freezing is a critical component of the technique.
- It has been successfully applied to study virus interactions.
- The method can be generalized to various applications in cell biology.
Purpose of Study
- To visualize virus-induced structures in cells.
- To enhance understanding of bio-protein interactions in cellular membranes.
- To provide a practical guide for researchers using this method.
Methods Used
- Preparation of patterned sapphire discs for imaging.
- Use of inverted microscopy for pattern confirmation.
- Sterilization of discs using ultraviolet crosslinking.
- Cell culture techniques for seeding cells onto sapphire discs.
Main Results
- Successful visualization of virus-induced intracellular structures.
- Demonstration of the effectiveness of CLEM in studying virus-host interactions.
- Practical steps outlined for implementing the method.
- Potential applications in broader cell biology contexts.
Conclusions
- The CLEM method provides valuable insights into virus biology.
- It bridges the gap between light and electron microscopy.
- Further applications in cell biology are encouraged.
Frequently Asked Questions
Executive Industry Relevance
Correlative Light Electron Microscopy (CLEM) addresses a critical bottleneck in virology research by enabling targeted analysis of rare transfected or infected cell populations. By combining fluorescence-based live-cell imaging with high-resolution ultrastructural preservation via high pressure freezing, this method enhances predictive confidence in mechanistic studies of virus-host interactions. It supports early-stage target validation and assay development by providing correlative data that bridges functional phenotypes with subcellular architecture, informing go/no-go decisions in antiviral discovery pipelines.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by linking fluorescently tagged viral protein expression to ultrastructural changes in cryo-immobilized cells.
- Operational Value: Increases efficiency in studying low-efficiency transfection or infection events through pre-screening via light microscopy.
- Predictive Value: Supports mechanistic de-risking by visualizing virus-induced membrane alterations and assembly sites relevant to replication complexes.
Screening & Assay Development
- Scientific Value: Generates correlated fluorescence and electron density readouts that enable quantitative assessment of viral protein localization and structural perturbations.
- Operational Value: Standardizes sample preparation using patterned sapphire discs, facilitating reproducible correlation across experiments and users.
- Assay Readiness: Produces ultrastructurally preserved samples suitable for downstream imaging and morphometric analysis in screening campaigns.
Translational & Preclinical Research
- Translational Continuity: Maintains structural fidelity from live-cell imaging to electron microscopy, supporting biomarker-aligned phenotypic screening in disease-relevant systems.
- Preclinical Modeling: Enables detailed characterization of virus-induced cytopathic effects in near-native states, improving confidence in translational extrapolation.
- Risk-Adjusted Advancement: Provides ultrastructural evidence to support or refute mechanisms of action for antiviral candidates.
Pipeline & Workflow Integration
CLEM fits within the discovery continuum from hypothesis generation in early virology to mechanistic validation in lead optimization, particularly when structural correlates of protein function are needed.
- Discovery Biology: Supports hypothesis testing by enabling direct visualization of viral protein-associated structures in defined cellular subpopulations.
- Screening: Enhances assay reliability through standardized cell localization and cryo-preservation, reducing variability in structural readouts.
- Analytics: Delivers correlated light and electron microscopy datasets that allow quantitative comparison of conditions based on fluorescence intensity and ultrastructural features.
- Translational Research: Bridges functional imaging with high-resolution structural data, supporting continuity from cell-based assays to mechanistic validation.
- Enterprise Reuse: Establishes a reusable platform for studying virus-host interactions applicable across multiple targets and viral families.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity by providing direct correlative evidence of viral protein localization and induced structural changes.
- Operational Value: Improves reproducibility through standardized use of patterned substrates and high pressure freezing, minimizing artifacts from chemical fixation.
- Strategic Value: Informs better go/no-go decisions by validating target engagement at the ultrastructural level, reducing late-stage failure risk.
- Portfolio Impact: Enables risk-adjusted prioritization of antiviral candidates based on confirmed effects on viral replication compartments or assembly sites.
Implementation Considerations
- Requires expertise in live-cell fluorescence imaging, high pressure freezing, freeze substitution, and transmission electron microscopy.
- Dependent on access to high pressure freezers, ultramicrotomes, and correlated light and electron microscopy imaging systems.
- Necessitates standardization of fiducial markers (e.g., patterned sapphire discs) and coordinate mapping protocols across teams.
- Adaptation to different cell types or viral systems may require optimization of transfection efficiency, fluorescence labeling, and freezing parameters.
- Practical limitations include low throughput due to specialized equipment and the need for correlative software alignment, as noted in the source material.
Biopharma FAQs
Chapters in this video
0:04
Title
0:51
Patterned Sapphire Disc Preparation, Cell Culture, and Imaging
3:16
Cryo-immobilization and Freeze Substitution (FS)
5:24
Resin Embedding and Patterned Sapphire Disc Removal from the Polymerized Resin Block
6:43
Ultrathin Section Preparation and Imaging via Transmission Electron Microscopy (TEM)
8:06
Results: Tracking and Imaging of Viral Protein-associated Structures via Correlative Light Electron Microscopy (CLEM)
8:47
Conclusion
