How does a cryo-EM microscope work?

Cryo-electron microscopy, or cryo-EM, is an important analytical method in structural biology. It enables researchers to determine the three-dimensional structures of biological macromolecules by, first, flash-freezing them in a vitreous (glass-like) ice state. This preserves their native conformation without the need for crystallization or staining. Frozen samples are then imaged using transmisson electron microscopy (TEM). Sophisticated computational techniques are then used reconstruct a 3D density map from that TEM data. Thus, researchers can visualize biological macromolecules in near-native states.

Here’s an in-depth look at the key components and technological advancements that make cryo-EM a groundbreaking tool in structural biology.

Sample Preparation

Sample prep is the first step in cryo-EM. It involves the rapid freezing of samples in liquid ethane or a similar cryogen to create a vitreous ice layer that embeds the sample. This vitrification process prevents ice crystal formation, which could damage the sample’s structure. Freezing samples quickly is key for preserving the specimen in a hydrated state that closely mimics their natural environment. Traditional electron microscopy uses staining, or samples may be embedded in plastic, to preserve a biological sample’s natural conformation.

Electron Microscopy

TEM is used to image frozen samples in cryo-EM. In a TEM, electrons are accelerated and focused onto the sample using magnetic fields. As electrons pass through the sample, they interact with the sample, and change in the phase of the electron wave carries information about the sample’s structure. The electrons that pass through are then focused onto a detector, forming an image.

Recent advancements in TEM technology, especially the development of direct electron detectors, have significantly improved the resolution and quality of cryo-EM images. These detectors are faster, more sensitive to the electrons and provide higher signal-to-noise ratios, enabling the visualization of finer structural details.

Image Processing and 3D Reconstruction

The raw images obtained from TEM, known as micrographs, are two-dimensional projections of the sample. To reconstruct the three-dimensional structure, thousands to hundreds of thousands of images of individual particles are extracted from the micrographs. Advanced computational algorithms are used to classify the images into groups representing different views or conformations of the molecule.  These images are then aligned and averaged to improve signal-to-noise ratios. 3D models are reconstructed from these aligned class averages. The resolution of the final structure depends on the quality of the sample as well as quality and quantity of the images, and the effectiveness of the image processing software.

Recent software developments have introduced more sophisticated algorithms for image alignment, classification, and 3D reconstruction, enabling near-atomic resolution structures to be obtained.

Technological Advancements and Automation

The cryo-EM field has benefited from several technological advancements, including improvements in electron microscope hardware, automated data collection systems, and computational power. Automated procedures for data collection and processing have made it possible to handle the large datasets required for high-resolution work more efficiently. Additionally, developments in cryo-EM grid technology and sample handling systems have improved sample stability and image quality.

Interested in Cryo-EM Services?

Cryo-EM is a cutting-edge technology that provides high-resolution structural details comparable to X-ray crystallography without the need for crystals. This technique is essential for competitive research projects. It offers rapid and flexible access to cryo-EM capabilities for both proprietary and non-proprietary clients. 

At the Hauptman-Woodward Medical Research Institute, our facilities are equipped with state-of-the-art instrumentation, including the Thermo Fisher Glacios 200 kV microscope with advanced features like a Volta phase plate and a Falcon 4 Detector. The laboratories are purpose-built with specialized equipment for sample preparation and grid handling, ensuring the highest degree of success and confidentiality.

Our cryo-EM center boasts expert staff with over 30 years of combined experience, providing full support throughout the structural determination process. Users have timely access to experimental runs, with the option to prearrange sessions to accommodate specific sample requirements. Moreover, the center prioritizes confidentiality and security, leveraging decades of experience in structural biology and collaborations with pharmaceutical and biotechnology companies.

We offer rapid and flexible access to cryo-EM capabilities for proprietary and non-proprietary clients, together with expert guidance at competitive rates. Contact us to learn more about cryo-EM.

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