Cryo-Electron Microscopy

What it is, How it Works, Pros and Cons

What is Cryo-Electron Microscopy?

Essentially, Cryo-electron microscopy (Cryo-EM) is a type of transmission electron microscopy that allows for the specimen of interest to be viewed at cryogenic temperatures. Following years of improvement, the cryo-electron microscope has become a valuable tool for viewing and studying the structures of various biological molecules. 

*  Transmission Electron Microscopy (TEM) refers to a technique where the image (of specimen) is formed by directing a high energy electron beam at a thin sample


While cryo-electron microscopy encompasses a number of experimental methods (imaging intact tissue sections, imaging plunge frozen cells and virus etc), these methods are based on the principle of imaging radiation-sensitive specimens in a transmission electron microscope.

Understanding the Significance of Electrons (Electrons vs. Photons)

Photons are packets of energy (basic particles of light) therefore, everything that one sees with their own eyes is due to the fact that these particles reflect off the physical objects we perceive and into our eyes. However, because some of the objects (of specimen in this case) are too small compared to the wavelength of photons, they are unable to interact making it impossible to view them.

When it comes to the wavelength of electrons, it is small enough to interact with the objects making it possible to observe them. Therefore, electrons become more suitable for the purposes of observing the small components of cells as well as a variety of molecular structures.

Transmission Electron Microscope

There are two major types of electron microscopes; the scanning electron microscope and the transmission electron microscope. However, because of the fact that the transmission electron microscopes offer higher resolution compared to the scanning electron microscopes, they are preferred when it comes to structural biology.

The following are some of the major parts of TEMs:

  • The Electron Column - This is the part of the microscope that contain the electron source (produces electron beam), lenses that focus the electron beam as well as the imaging system where the image is projected
  • The Vacuum System - This is a component of the microscope that helps minimize collision frequency of electrons with gas atoms. As such, it enhances the efficiency of the electron gun

How the Cryo-EM Works

Transmission electron microscopes use the same working principle as the ordinary light microscope. However, rather than using the limited wavelength of light, electrons with much lower wavelengths are used as the source of light.

For the TEM, there are two types of electron sources that are commonly used. These include the thermionic electron guns and the field emission guns. Whereas electrons are emitted from such heated filaments as bent tungsten or sharp lanthanum hexaboride crystal in thermionic electron guns, they are emitted from a sharp, pointed cathode by a string electric field in field emission guns.

For TEM, the electron gun uses electronic coils as well as high voltages to accelerate the electrons to extremely high speeds (thus shorter waves). The electrons then travel through the anode, an aperture and into the vacuum tube.

Unlike the light microscope, the TEM has in place electromagnetic lenses that bend the electron beam though the Lorentz force. The lenses also direct the beam through the tube and onto the specimen.

Basically, the transmission electron microscope can be said to have three important systems.

These include:

  • The Electron Gun - The electron gun is the part of the microscope that is responsible for producing the electron beam. Here, the condenser system is responsible for focusing the electron beam on to the specimen sample.
  • Image Producing System - This is composed of the objective lens, intermediate and projector lenses as well as a movable stage. The lenses are also involved in the focusing of electrons, which helps form the magnified image.
  • Image Recording System - This part of the microscope is mostly composed of a fluorescent screen that helps in producing an image that can be seen with the eye. Here, a digital camera also helps capture the images for documentation.


In Cryo-Electron Microscopy the sample under observation is usually frozen (frozen-hydrated) for preservation purposes. Here, a very thin slide of the specimen may be rapidly plunged into a liquid ethane bath and viewed in their natural state. Solvents like water or a salt solution is used to ensure that the sample remains stable.


* The solvent water around the specimen is frozen in place when the sample is plunged into the cold medium thus cryogenically preserving and protecting the specimen.


Here, it is very important that the process of freezing the sample is very quick. This prevents the frozen water around the specimen sample from forming cubic ice. In the event that ice is formed, it readily absorbs the electron beam, which in turn obscures the sample. For this reason, it is essential that the sample be plunged in to the cooling liquid rapidly so that the water only freezes around the specimen for clear images.


While liquid nitrogen can also be used for the freezing process, ethane is used instead given that it has higher heat capacity and is also liquid at temperatures slightly above that of liquid nitrogen. As such, it is sufficiently cold to freeze water rapidly and appropriately without boiling off.

Sample Preparation

  • Fixation - This is the first and most important step of sample preparation for this technique. Here, the structure of the sample (tissue, cells etc.) has to remain as close to its natural form as possible.
  • The sample is then dehydrated using acetone/ethanol, passed through another transition solvent (such as propylene oxide) and ultimately infiltrated and embedded in such liquid resins as epoxy and LR white resin.
  • This is followed by sectioning of the sample. Sectioning if performed through ultramicrotomy to obtain sections of between 50 and 70 nm in thickness
  • Once collected on a metal mesh grid, the specimen can then be stained using electron dense stains


To observe macromolecules in their native hydrated state, the sample is simply embedded in vitrified water, frozen hydrated as directly visualized on the microscope. Here, no stains are used given that the surrounding buffer allows for enough contrast to observe the specimen.


Here, it is worth noting that different samples have different protocols. For this reason, it is important to confirm the right procedure for specific specimen.

Some of the main mounting techniques used in Cryo-EM include:


  • Surface Mounting  - used mostly for leaf specimens where the specimen is laid on top of the mounting media
  • Edge Mounting - Used for edge observation and fracture, edge mounting technique involves placing the specimen on its edge in a machine slot secured with a mounting media
  • Film Emulsion Mounting - This is used particularly in the event that the specimen is too small and can be obscured by the Tissue-Tek mounting media. Here, the specimen is laid on the surface allowing it to adhere to the film surface

Other techniques include:


  • Rivet mounting
  • Alternative rivet mounting method

Pros and Cons of Cryo-Electron Microscopy



Small Samples


One of the biggest advantages of cryo-electron microscopy is that very small samples are actually required for the determination of its structure. Compared to other microscopy techniques, cry-electron microscopy still produces good images (as long as the sample is in good condition).

A good example of this is with single-particle reconstruction which only requires 105 particles in order to reconstruct a near-atomic-resolution structure. Here, quality results can be expected as long as the sample of interest has good biochemical properties as well as high conformational homogeneity.

As for such samples as viruses which have high symmetry, only 104 particles would be required. On the other hand, a cryo-electron microscope specimen only requires between 3 and 5 ul of protein solution at a concentration of 1.2 to 1 umol L-1 which is in marked contrast compared to larger samples used in other techniques like crystallography and NMR spectroscopy.


Physiological State


The other significant advantage of this microscopy technique is that fixation of the sample involves rapid freezing and actual fixation in vitreous ice in order to preserve their hydrated state. Here, one gets to view a sample with structural information that to a large extent reflect the state of the sample before it was frozen.

With Cryo-EM, the sample under observation is in solution and does not come in to contact with any adhering surface. As a result, the shape observed is also the true shape (state) of the molecules given that the shape is not affected through attachments which can result in flattening.



This technique has an advantage in that it can be used to view and characterize a wide range of samples. Using cryo-electron microscopy, it becomes possible to view cells, cell organelles as well as macromolecules complexes of well over 500 kD.

Recently, Cryo-Electron Microscopy was also used to determine high resolution structures of 200kD proteins, which was a great achievement in the world of microscopy. Therefore, apart from only requiring small samples, this technique can serve to view study and characterize a wide range of specimen without the need to acquire various other devices.

Inhomogeneous Samples


Cryo-EM offers a great advantage in that it provides high magnification allowing for the specimen to be viewed and studied closely. Moreover, it offers a significant advantage in that through the direct acquisition of the images; the specimen can be statistically analyzed allowing for the reconstruction of the structural information.

Here, it becomes possible to classify different possible molecular structures from similar samples to other molecules with different conformations and compositions. Furthermore, the classification of a large number of single molecular structures also provides statistical distribution of different states.


Some of the other benefits of Cryo-Electron Microscopy include:

  • Stains are not necessary which means that the specimen is not distorted through the use of stains and other dyes. Given that low dose methods are commonly used, the electron beam does not cause much damage to the specimen.
  • It is possible to distinguish between nucleic acids, proteins and lipids.
  • It is possible to control the chemical environment, which in turn allows for effective examination of different functional states of different types of molecules.


Very low signal to noise ratio - This is one of the biggest disadvantages with Cryo-EM. For biological macromolecules, the main building blocks include carbon, hydrogen, oxygen and nitrogen. Given that electron absorption of these molecules is very low, then there is low contrast of the resulting images which makes it difficult to detect features of a given sample when viewing a few samples.

Although Cryo-EM offers great overall magnification, the issues with image contrast may make it difficult for some users (particularly new users) to distinguish between what they are viewing.

Tilt Imaging - With Cryo-EM, it is not easy to obtain images from a tilted specimen due to the cross section of the frozen sample. Here, the cross section of the ice is usually too thick making it difficult to obtain good images. For this reason, users have to ensure that the specimen is not tilted in order to obtain better images.

Here, this technique requires that the user take every step seriously or risk starting all over again. When imaging a tilted frozen sample, there is also another disadvantage of more widespread charging.


It's important to ensure that cubic ice is not formed during freezing. This is due to the fact that they absorb electrons making the sample worthless. As mentioned, this technique is very sensitive (particularly with sample preparation) which can be frustrating for some users.

Some of the other challenges with cryo-electron microscopy include:

  • It takes time to generate the sample
  • Fully hydrated specimen have been shown to be electron-beam sensitive
  • It's costly


While it has its challenges, cryo-electron microscopy has proved to be a valuable tool in various biological fields including botany, biotechnology and zoology among others. It addition, it is also being increasingly used in other industries such as pharmaceuticals, healthcare as well as cosmetics to name a few.

Because it can overcome a number of limitations that face other microscope techniques like light microscopy and scanning electron microscopy, Cryo-EM has allowed scientists and technicians to observe and study a wide range of molecules and their structures.

With improvements in the few coming years, the problems of Cryo-EM will corrected to make it one of the best tools in biology, medicine and research.

Check out a great page on Nanotechnology here 

Scanning (SEM) - Learn about the SEMs high-resolution, three-dimensional images which provide topographical, morphological and compositional information making them invaluable in a variety of science and industry applications. 

Transmission (TEM) - check out one of the most powerful microscopic tools available to-date, capable of producing high-resolution, detailed images 1 nanometer in size. 

Cryo-Electron - is a type of transmission electron microscopy that allows for the specimen of interest to be viewed at cryogenic temperatures.  Check it out.

Virtual - provides a simulated microscope experience via a computer program or Internet website for both educational and industrial applications and are easily operated and accessible. 

Take a look at how Electron Microscopy compares to Super-Resolution Microscopy

Taking a look at Viruses under the Microscope and answering the questionwhat are viruses?

As well as Atom under the Microscope and DNA under the Microscope

Electron microscopy (SEM and TEM) images of SARS-CoV-2 - Covid 19

DNA under the Microscope

Continued Reading:

Cryo-Electron Tomography

Electron Microscope

See Virus under the Microscope 

Return from Cryo-Electron Microscopy to MicroscopeMaster Home


Allison Doerr (2016) Single-particle cryo-electron microscopy.


Jacqueline L. S. Milne et al., (2013) Cryo-electron microscopy: A primer for the non-microscopist.


Richard Ellis Ford Matthews, Roger Hull (2001) Matthews' Plant Virology.


Wang HongWei (2014) Cryo-electron microscopy for structural biology: current status

and future perspectives.



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