Table of Contents
Introduction to Cryo Electron Microscopy (Cryo-EM):
Cryo Electron Microscopy (Cryo-EM) is a technique used to study the structure of biological macromolecules, such as proteins and nucleic acids, at high resolution.Cryo-EM uses a cryogenic electron microscope to visualize the samples in a frozen, hydrated state. This allows for the preservation of the native state of the molecule and the avoidance of radiation damage.
Sample Preparation for Cryo Electron Microscopy (Cryo-EM):
- Sample preparation is a critical step in Cryo-EM.
- The sample is typically vitrified, or rapidly frozen, in liquid ethane to preserve its native state.
- The sample is then placed on a thin carbon film on a electron microscope grid, which serves as a support for the sample and allows the electrons to pass through.
Microscope for Cryo Electron Microscopy (Cryo-EM):
- Cryo-EM uses a cryogenic electron microscope, which operates at temperatures close to liquid nitrogen (-196 ยฐC) to minimize radiation damage.
- The microscope uses a high energy electron beam to generate images of the sample.
- The electrons are scattered by the sample and the resulting diffraction patterns are captured by a detector to generate the image.
Data Collection and Processing:
- Data collection in Cryo-EM is typically done in a “single-particle” mode, where thousands of images of identical, but randomly oriented, particles are collected.
- The data is then processed using image processing software to align and average the images, resulting in a high-resolution, three-dimensional reconstruction of the sample.
- Single Particle versus Cryo-tomography:
- Cryo-EM can be done in two ways: single particle mode and cryo-tomography.
- Single particle mode is used to determine the structure of individual macromolecules, while cryo-tomography is used to study the three-dimensional organization of macromolecules within a cell.
Resolution and Limitations:
- Cryo-EM is capable of producing high-resolution structures, with resolutions reaching near atomic level.
- However, the resolution is dependent on the quality of the sample and the size of the macromolecule being studied.
- Furthermore, Cryo-EM is limited by the need for a sufficient number of identical particles in the sample, which can be difficult to obtain for some macromolecules.
Conclusion:
Cryo Electron Microscopy (Cryo-EM) is a powerful technique for studying the structure of biological macromolecules at high resolution. The technique uses a cryogenic electron microscope and a sample that is rapidly frozen to preserve its native state. Cryo-EM can be done in single particle mode or cryo-tomography mode depending on the research question. It has the potential to reach atomic resolution but it is limited by the need for a sufficient number of identical particles and the quality of the sample.