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
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
