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 °C) 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.