What is CryoEM?
Cryo-electron microscopy (also known as electron cryomicroscopy or cryoEM) is the method our lab uses to “take photographs” of viruses and other macro-molecular complexes. The following is an abbreviated, layman’s terms explanation of how cryoEM works.
First, we must prepare the specimen for studying with the electron microscope. To image a virus, we start by growing the virus to a high titer (a high concentration), isolating it, and then purifying it. In order to observe the virus sample in the microscope, we first must place it on a small circular physically-supportive platform, called a grid. A drop of the purified sample (which contains thousands of virions) is placed onto a grid, and then the grid is quickly frozen to -180 degrees Celsius using liquid nitrogen and liquid ethane.This freezing procedure is done in order to protect and preserve the specimen while it is being observed under the microscope.
When the sample is ready, we can start shooting electrons at it. Unlike light microscopes, which use glass lenses to focus light onto a sample, electron microscopes use magnetic coils (magnetic lens) to accelerate and focus electrons onto a sample. The shape of a sample can become destroyed and distorted by prolonged exposure to electrons. Since the objective of cryoEM is to observe the natural structure of a biological sample with minimal distortion, cryoEM uses a very low dose of electrons (about 1-10 e‾ per Å²) while imaging samples. Electrons that pass through empty areas result in dark areas and electrons that bounce or refract from dense areas result in light areas.
When imaging is complete, we end up with a black and white 2D image similar to the one in the diagram on the right. We collect thousands of these 2D images, each of which should ideally contain a different perspective of the sample (i.e. top, bottom, side, etc.). Collecting images of the sample from various angles is necessary for us to fully understand how the sample looks in 3D space. This collection of flat 2D images is combined and rendered into 3D model of the sample using computerized 3D data merging software. More information about the 3D data merging process can be found here:
The final result is a 3D model of the biological sample. This model can then be used to expand our understanding of biological mechanisms and to inform health-related research, such as drug-targeting and vaccine development.
What is CryoET?
Cryo-electron tomography (cryoET) is an imaging method similar to cryo-electron microscopy. Both cryoET and cryoEM require acquiring multiple perspectives of the biological sample in order to compile a 3D model of the sample. However, the number of samples used for each method differ.
- CryoEM takes pictures of an array of many different particles that are frozen in various orientations (i.e. face-up, face-down, tilted to the side, etc.).
- CryoET takes sequential pictures of a single particle as it is slowly rotated (or tilted) along one axis.
To illustrate the difference between cryoEM and cryoET with a simplified example, consider a 6-sided die. If we wanted to create a 3D model of the die, we would need to acquire pictures of each of the 6 differently numbered sides (1, 2, 3, 4, 5, 6). There are 2 main ways to do this:
- Roll 100 dice (or more), and expect that all 6 numbers have been rolled at least once each (CryoEM)
- Take 1 die and rotate it in your hand in order to see all 6 sides (CryoET)
Both methods would result in a collection of pictures of the sample (the 6-sided die) from all 6 possible perspectives.
Although cryoET is useful for creating 3D models for structures that are specific or unique to a single particle, and for flexible samples that are difficult to visualize with cryoEM, there are drawbacks as well. Since cryoET imaging constantly exposes a single particle to radiation damage for the entire duration of the imaging session, the structural integrity of the sample is more quickly degraded than in cryoEM and the number of images that can be taken is limited.