How Cryo-EM Differs from TEM

Cryo-electron microscopy (cryo-em) and transmission electron microscopy (TEM) share many similarities, but their main difference is in sample prep.


How Cryo-EM Differs from TEM

Transmission Electron Microscopy or TEM is a generalized term for a suite of imaging techniques that uses electrons to acquire image data and/or analytical data from a specimen.  Cryo-electron microscopy or cryo-EM, a de facto sub-set of TEM, has as a goal acquiring information from biological samples whose native state has been preserved whilst in the vacuum of the electron microscope. Cryo-EM has yielded a surge of near- and real atomic structures solved for instance by single particle analysis (SPA) or sub-tomogram averaging, all deposited in the structure data banks around the world. 

As alluded to above, the key difference between cryo-EM and conventional TEM is the sample preparation method. Cryo-EM uses flash or slam or jet freezing of a liquid or suspension to create a specimen that can be observed in the microscope without the use of fixating or staining aids, whereas conventional TEM methods typically include approaches like chemical fixation or staining agents or even the use of polymers to immobilize the sample in place.1 This principal difference in sample preparation dictates what types of specimens can be studied by either technique. Conventional TEM has been widely used in cellular imaging for over fifty years and can reveal information on the structure of cell organelles.2 This approach is ideal for revealing ultrastructural elements e.g., cellular components, vesicles, ER etc, albeit with limited resolution. Cryo-EM, on the other hand, in avoiding the fixation and staining preserves the structure of macromolecular complexes to high resolution, which can be retrieved using SPA or sub-tomogram averaging applied to tomography data. Also, and more critically important, cryo-EM allows us to examine those complexes that are typically not amenable to other structural studies, such as NMR or x-ray crystallography, because either the complexes are simply too large or crystals are impossible to grow. Furthermore, cryo-EM can address those cases where the complex of interest is available in only minute quantities, such as reaction intermediaries, or the complex is present in a conformational mixture. The advent of direct detectors, highly automated electron microscopes and powerful image processing algorithms, responsible for the resolution revolution, have established cryo-EM as an increasingly popular tool in structural biology projected to eclipse x-ray crystallography as the technique to solve atomic structures in a few years (Kikkawa, 2022).

In-situ TEM, where biological samples are imaged in a liquid cell at room temperature, has the potential to yield information regarding fluctuations in the protein structure as a function of temperature.3 The level of detail, however, may be limited to domains owing to the vast increase in radiation sensitivity of samples studied by this technique, and the possible mobility of a complex in solution. On the other hand, recent computational approaches applied to cryo-EM data sets of ribosomes obtained by SPA have suggested various conformational states that were observed with substantially greater detail (cryoDRGN, 2022).


The key components of the instrumentation for cryo-EM and TEM can be the same. Both approaches require an electron source – typically an electron gun capable of producing electron beams with different energies but whose characteristics depend critically on the type of source employed.
The electron beam must then be focused onto the sample using a series of lenses, which occurs in the condenser system. These lenses help to shape the beam and achieve the small spot sizes necessary for obtaining the best spatial resolution with the electron beam as well as achieve the proper dose rate for various studies.
The heart of the electron microscope is the objective lens. This optics piece, composed of an upper and lower pole piece, is responsible for the level of detail observed in an electron microscope. Worth noting is that owing to the presence of spherical aberration, high-resolution images can be acquired from unstained cryo-EM samples by simply defocusing the objective lens, typically by a fraction of a micron.
Inserted in the gap of the objective lens is the sample holder. For automated, high-throughput studies an autoloader is paramount, whereas a side-entry holder suffices for other cases. Imaging cryo-EM samples is principally done using a stationary beam. In Materials TEM, the preferred technique is to raster the electron beam across the sample.
Below the sample are magnifying lenses and finally, at the very bottom of the column, is typically a direct electron detector or DED. Largely responsible for the resolution revolution in cryo-EM, DEDs record movies of the sample rather than still images, thus providing for a way to eliminate beam-induced motion. Typical cameras in conventional TEM do not require this and are thus far cheaper and simpler.

JEOL Microscopes

Performing cryo-EM has never been so easy as with the JEOL CRYO ARM 300 II. With the capability of performing SPA and tomography for 3D reconstructions, the CRYO ARM 300 II makes it simple to harness the excellent spatial resolution and structure-solving capabilities of cryo-EM.
Contact JEOL today to find out if the CRYO ARM 300 II could be the instrument for you and how cryo-em and advanced TEM methods could benefit your application. JEOL offers a full suite of Transmission Electron Microscopes, with unique options such as the 120kV JEM-1400Flash, the LaB6 JEM-2100, and more.


  1. Nagashima, K., Zheng, J., Parmiter, D., & Patri, A. K. (2011). Biological Tissue and Cell Culture Specimen Preparation for TEM Nanoparticle Characterization. In S. E. McNeil (Ed.), Characterization of Nanoparticles Intended for Drug Delivery (pp. 83–91). Humana Press.
  2. Winey, M., Meehl, J. B., Toole, E. T. O., Giddings, T. H., & Drubin, D. G. (2014). Conventional transmission electron microscopy. Molecular Biology of the Cell, 25, 319.
  3. Nagashima, K., Zheng, J., Parmiter, D., & Patri, A. K. (2011). Biological Tissue and Cell Culture Specimen Preparation for TEM Nanoparticle Characterization. In S. E. McNeil (Ed.), Characterization of Nanoparticles Intended for Drug Delivery (pp. 83–91). Humana Press.


Cryo-EM, TEM



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