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Cryo Electron Microscopy
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The Emergence of Path Breaking Cryo-Electron Microscopy

In the early 1930s, Ernst Ruska and Max Knoll, a physicist and an electrical engineer, respectively, from the University of Berlin, invented the first Electron Microscope. The electron microscope uses an electron beam to produce an image of the object, but somehow electron beam disfigure the object, so to overcome these problem scientists started working towards totally new approach and invented Cryo-EM in the 1980s.

Shaping the path for ‘Nobel Prize’

In 1975, Joachim Frank started working on algorithms of Cryo-EM wherein the 2D images were arranged in 3D structure. In the early 1980s, Jacques Dubochet, understood how biomolecules can retain their original shape with the help of vitrifying water. Lastly, in 1990, the first electron microscope was used by Richard Henderson to generate a 3D structure of a protein. After all these inventions, the trio of scientists Jacques Dubochet, Joachim Frank and Richard Henderson were awarded nobel prize in 2017 by Royal Swedish Academy of Sciences for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in the solution.

Diving deep to understand Electron Microscopy

There exist two variants of Electron microscope – Transmission Electron Microscope (TEM) and Scanning Electron Microscope (SEM).

TEM works under the principle that, an electron beam passes through the sample and generates a fluorescent image. Whereas, In SEM electron beam scans over the surface of the sample and creates an image.

Cryo-EM follows the principle of TEM, it uses frozen samples, electron beams and generates a sophisticated 3D image. It makes use of electrons beam for the examination of the structures whether it is a cellular, virus or at the atomic scale. When the beam passes through the sample it interacts with the molecules and this, in turn, produces an image onto the detector. Usually, a charge-couple device is used as a detector. Finer detailed imaging of the sample is produced due to the wavelengths of the electrons being shorter than that of the light.

Three Dimensional Sample Image

The first step in Cryo-EM is the process of vitrification. In this process, the sample is first rapidly frozen so that the water molecules form an amorphous solid instead of crystallizing to avoid damage to the sample as far as possible. After this, the sample is screened for concentration, distribution, and orientation of the particle. A series of images are assimilated and 2D- images of them are extracted computationally. The last step in the procedure is to process the data by reconstruction software to give detailed 3D models of the cellular structures.

The key to scientific results is that these models can reveal interactions in the structures that were not visually possible before.

Looking at the procedure

Two methods are used for sample preparation, one is a thin film – wherein the specimen is placed in an EM grid and rapidly frozen without crystallizing it and second, vitreous water – wherein the larger samples are vitrified by high-pressure freezing, cut thinly and placed on EM grid.

Sample Preparation:

Vitrification – It is necessary to avoid the formation of ice, this state is maintained by using liquid nitrogen. The sample under assessment is positioned on a carbon grid and immersed into the ethane bath kept inside a liquid nitrogen container. After the vitrification of the sample, it is cut into a small section using diamond knives. The grid on which sample is placed made up of carbon.

Observation of specimen – The variance of the specimen depends on the specimen itself, defocus value of the objective lens and thickness of the ice. By using a fluorescent screen and the photographic film, we can observe the image.

3D reconstruction –Raw images from different projections are recorded and they are stitched together to form the required 3D structures using computer software.

Cryo-EM transforming the scientific world

The Cryo-EM method has been used in various fields to analyze structures in high resolution which other methods are not capable of.

One of the areas this technique has been used is to study T4 bacteriophage. Its structure, assembly, and interactions were studied in a three-dimensional phase. A very promising aspect of the Cryo-EM technique is time-resolved imaging. This type of imaging allows snapshots to be taken of the macromolecular complexes in a reaction or a transition. This approach has been used to study isomers of the T4 capsid of the bacteriophage.

It is also being used to study virus-infected cells by using tomographic reconstructions which allows us to see the life cycle of a virus in the infected cell.

Cryo-EM has allowed researchers to study and define the world’s first protein structure know as human Ataxia Telangiectasia Mutated (ATM). The studies on this allow us to understand the DNA damage response and use it to understand the depth of cancer research.

The ternary complex structure of the calcitonin structure has been studied due to the use of Cryo-EM.

Path-breaking developments using Cryo-EM

Cryo-EM has revolutionized in recent years.  One of them is the study of large protein complexes which can directly be done by applying on the grid now and by avoiding the crystallization step. Another recent development was to analyze an image of Adenovirus particles providing resolution for tracing the polypeptide chains in the capsid structure. This was done by achieving a high resolution of approximately 3.5 Angstrom. Due to the recent advances in the resolution of imaging, it has also been possible to put Cryo-EM to use in the field of drug discovery.

The signal to noise ratio in a Cryo-EM is quite poor. To improve this, the use of phase plates have been considered and are considered an emerging technology in Cryo-EM. These phase plates allow viewing of cellular structures in finer detail and for Single Particle Analysis [SPA], the phase plates allow the study of small molecules and complexes.

Correlative Light and Electron Microscopy [CLEM] is being used to analyze rare events occurring in cryopreserved samples. Prediction of the position of molecules in 3D using Cryo-CLEM has shown to give a precise image up to 200nm.

Cryo-FIB or Cryo-focused ion beam has been used to solve the issue of sample preparation and it is possible now wherever needed to obtain a thinner sample.

Advantage of using Cryo-EM:

  • It allows the observation of a specimen that has not been stained, which is very useful to determine the structure and function of macromolecules.
  • Showing specimen in their native state – Sample stays in the solution so that it doesn’t come in contact with an adhering surface, due to this it is observed in its true shape.
  • Allows to view very fine structures in small organisms such as viruses and bacteria, cellular parts and protein complexes that require molecular resolution
  • Provides atomic resolution of the protein complexes
  • Automated 3D construction and much more

Way Ahead

Cryo-Electron Microscopy is a form of Transmission Electron Microscopy which has allowed us to obtain new insights into the cellular structure which could not be observed by using other methods. Biological molecules can be visualized in 3D images and in its native form. There are certain limitations to this method which majorly revolves around the thickness of the sample along with resolution not being high enough. Recently advancements towards higher resolution have been achieved along with obtaining thinner samples for the experiments. Continuous advancements will eventually lead to better imaging and sample preparation method. This method continues to improve and eventually will become more popular among many cell biologists. The recent use of Cryo-EM for deciphering CRISPR-Cas9 image has marked an era of revolution in the scientific field. Let’s look ahead for more such developments to make the understanding of science more comprehensive.

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