Life is composed of cells with several magnitudes smaller than a grain of salt. Although their structures appear simple, they can carry out complex molecular functions that sustain life.

It is becoming easier for researchers to visualise this activity at a level of detail they have never been able to before. It is possible to imagine biological structures either by starting at the level of the whole organism and working downwards or at the level of single atoms and working upwards.

Nevertheless, there is a resolution gap between minor structures of a cell, such as the cytoskeleton, and its most significant structures, such as the ribosomes, which produce proteins.

Like Google Maps, scientists can see entire cities and individual homes, but not how the houses come together to form neighbourhoods. Understanding how components work together in a cell’s environment requires seeing these neighbourhood-level details.

An overview of microscopy

Cells were first discovered by light microscopy in the 17th century [1]. With electron microscopy in the 20th century, cell structures were revealed with even greater detail, including organelles like the endoplasmic reticulum, which plays a key role in protein transport and synthesis.

During the 1940s and 1960s, biochemists studied the 3D structures of proteins and macromolecules at or near atomic resolution by separating cells into their molecular components. In order to visualise the structure of myoglobin, a protein that supplies oxygen to muscles, X-ray crystallography was first used.

In the past decade, nuclear magnetic resonance and cryo-electron microscopy have rapidly increased the number and complexity of structures scientists can visualise [2].

Cryo-electron tomography

New tools are steadily bridging this gap. As a result, cryo-electron tomography, or cryo-ET (CET), can deepen researchers’ understanding of how cells function in health and disease [3].

Biological specimens in their native, hydrated state can be imaged at high resolution using cryo-electron tomography. For example, it can be used to study the structure of viruses, organelles, and cells in three dimensions (3D).

In CET, a thin section of the specimen is frozen in vitreous ice and tilted images are taken from different angles using an electron microscope. The images are then computationally reconstructed to create a 3D model.

Comparing the cryo-EM and cryo-ET techniques

In cryo-electron microscopy, a camera is used to detect how electrons are deflected as they pass through a sample in order to see molecular structures. Radiation damage is prevented by rapidly freezing samples. By averaging multiple images of individual molecules into a 3D structure, detailed models of the structure of interest can be formed.

Despite having similar components, cryo-ET uses different methods than cryo-EM. Due to the thick nature of most cells, a region of interest in a cell must first be thinned with an ion beam [4]. As with CT scans of body parts, the sample is tilted to take multiple images at different angles, but with an imaging system instead of the patient tilted. 

These images are then merged by a computer to create a 3D image of the cell. In this image, researchers or computer software – can distinguish the individual components of different structures in a cell due to its high resolution. As an example, researchers have used this approach to show how proteins circulate and are degraded in algal cells.

Scientists can now identify new structures at an incredible speed because many of the steps they used to do manually are becoming automated. By combining cryo-EM with artificial intelligence programs such as AlphaFold, for instance, new protein structures can be predicted that have not yet been identified [5].

Advantages and limits of using CET

One of the major advantages of CET is that it allows researchers to study biological specimens in their native, hydrated state, without the need for chemical fixation or dehydration. This is important because chemical fixation and dehydration can alter the structure and function of the specimen, leading to artifacts in the final image.

CET is also capable of achieving a high level of resolution, on the order of 1-2 nanometers. This allows researchers to study the fine details of biological structures and interactions, and to identify specific proteins and other biomolecules within the specimen.

One limitation of CET is that it requires a high-vacuum environment, which can be challenging for certain types of specimens. Additionally, the technique is relatively time-consuming, requiring specialised equipment and trained personnel to operate it.

Despite these limitations, CET has become an increasingly popular tool for studying the 3D structure of biological specimens, and has contributed to numerous important discoveries in the field of structural biology. For example, CET has been used to study the structure and function of various viruses, such as HIV and influenza and to investigate the organisation of proteins within cells [6, 7, 8]

Overall, CET is a valuable tool for studying the 3D structure of biological specimens, and has the capability to provide crucial insights into the functioning of cells and the mechanisms underlying disease.

Structure and function of cells

Different strategies will be available for tackling some key questions in cell biology as imaging methods and workflows improve. Choosing which cells and regions within those cells to study is the first step. CLEM is another method of visualising living cells by using fluorescent tags to locate regions of interest [9].

In the same way that understanding how complex systems work becomes easier when you know what they look like, knowing how biological structures fit together in a cell is crucial to understanding how organisms work [10].

[1] https://doi.org/10.1098/rsob.150019
[2] https://www.nobelprize.org/prizes/chemistry/2002/press-release/  
[3] https://doi.org/10.1002/1873-3468.13948
[4] https://doi.org/10.1038/nmeth.4115
[5] https://doi.org/10.1038/s41586-021-03819-2
[6] https://pubmed.ncbi.nlm.nih.gov/31332385/
[7] https://pubmed.ncbi.nlm.nih.gov/24506064/
[8] https://pubmed.ncbi.nlm.nih.gov/25901680/
[9] https://doi.org/10.1002/1873-3468.14421
[10] https://www.sciencealert.com/this-technique-is-revealing-a-hidden-world-of-biology-weve-never-seen-before