From Ice Contamination to Carbon Artifacts: A Guide to Understanding & Overcoming TEM Imaging Challenges

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February 6, 2024

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B&W microscopic Cryo-TEM images of nanoparticle formulations

Understanding Common Artifacts observed  in Transmission Electron Microscopy Images

Even when working with an ideal sample that shows good particle concentration, distribution, and contrast within the ice or stain, experimental artifacts and grid imperfections can make acquiring a useful cryo transmission electron microscopy (cryo-TEM) or negative stain TEM dataset challenging.

Common artifacts that stem from grid preparation or substrate can potentially be present throughout all nanoparticle sample types, including liposomes, lipid nanoparticles (LNPs), viruses, virus-like particles (VLPs), exosomes, iron nanoparticles, and beyond, and are not necessarily inherently related to the sample. 

We polled NIS scientists, to find which commonly seen artifacts are the most troublesome, and the results are:

  1. Crystalline Ice Contaminants
  2. Stain Crystal Clusters
  3. Carbon Artifacts
  4. Drift
  5. Grid Imperfections

While some minor experimental artifacts are to be expected, NIS scientists have techniques to overcome each obstacle in order to provide quality TEM images and data. 

Crystalline Ice Contaminants in Cryo-TEM Images

NIS scientists unanimously agreed that excessive contamination of grids by crystalline forms of ice is the top challenge when acquiring quality images.

The vitreous ice necessary for cryo-TEM imaging is formed by rapid freezing of a thin layer of sample solution spread over a TEM grid into a cryogen such as liquid ethane. The high cooling rate turns the water in the sample into a non-crystalline, amorphous state that is electron transparent. This prevents the formation of ice crystals, preserving the particles in their native hydrated environment. The vitrified ice layer containing the particles must be kept near liquid nitrogen temperature (-196 C) at all stages to prevent unwanted ice types that may reduce image quality. But when the grid preparation or grid loading process does not go perfectly and excessive crystalline ice is formed on the grid, cryo-TEM can quickly become cry-TEM.

Microscopic black and white image of cryo-TEM imaging | nanoparticle characterization at nanoimaging services
 Left: Ideal cryo-TEM image of lipid nanoparticles (LNPs) Right: An instance of “cry-TEM” with extreme ice contamination obscuring LNPs.

Crystalline ice forms can be introduced during several steps of the sample vitrification process:

  • The grid may become contaminated by ice crystals present in the liquid nitrogen used to load the grid into the microscope. This is reduced by using liquid nitrogen that has been freshly dispensed from the tank.
  • Contamination of the grid by ice crystals which form due to water vapor present in the air. This source of contamination can be prevented by preparing grids in a dehumidified environment and by wearing masks while handling grids.
  • The initial sample solution layer on the grid may be too thick, resulting in freezing that is slow enough to form crystalline ice. This is remedied by optimizing the vitrification parameters, including blotting time.
  • While remaining frozen, the grid may warm slightly resulting in formation of crystalline ice. This is preventable by pre-cooling all tools and loading components that touch the grid. 

Ice contaminants on the grid vary widely in size and appearance, but always appear more dense compared to the thin sheet of vitreous ice and the embedded sample particles.

Microscopic black and white image of cryo-TEM crystalline ice contamination imaging | nanoparticle characterization at nanoimaging services
The poor quality images above show different forms of crystalline ice. Left: Ice contaminants of various sizes (green arrows) surround and overlap a lipid nanoparticle (LNP) (pink circle). Right: Signs of crystalline ice (green arrows) that obscures an LNP (pink circle).
Microscopic black and white image of cryo-TEM crystalline ice contamination imaging | nanoparticle characterization at nanoimaging services
Ice contaminants range from small (green arrows) to large (blue arrow) and take on a variety of shapes or cluster (purple arrow). In the image to the right the large ice contaminants are merely an aesthetic issue as they do not cover the particles within the hole.

A small amount of ice contamination is typical. If there’s excessive ice contamination, however, it can interfere with image interpretation and analysis. 

As long as the level of ice contamination is minor, single particle analysis is somewhat tolerant of these contaminants, as they won’t affect 2D classification and 3D structure reconstructions of the much larger number of particles of interest. 

Characterization of nanoparticles is more easily affected by ice contamination due to the oftentimes lower number of particles present on the grid. If the ice contaminants cover or obscure the particles, it can interfere with particle sizing, morphology and/or payload analysis, and image interpretation.

In some cases, ice contamination is less prevalent in certain areas of the grid, and data can be collected in that area instead. If ice contamination is prevalent across the entire grid, the grid is remade until a suitable one is found that allows for collection of quality of cryo-TEM data.

Stain Crystal Clusters Obscuring Particles in Negative Stain TEM

The second most troublesome image artifact occurs during negative stain transmission electron microscopy (TEM).

Negative staining of nanoparticles provides images with excellent contrast by surrounding the relatively electron transparent particles with an electron dense heavy metal solution. Uranyl formate, uranyl acetate, and phosphotungstic acid are commonly used to visualize proteins, viruses, or virus like particles (VLPs) with negative stain TEM. 

The ideal outcome for negative staining is an even dark background against which bright particles stand out. However, sometimes the background may be uneven due to the presence of clusters of stain crystals. Some amount of non-ideal staining can be worked around by targeting only areas with even stain. If this is not possible, and the stain clusters prevent clear views of a significant number of particles, another grid will be made. Sometimes, the presence of stain clusters is due to interaction between the sample buffer and stain during the process of grid preparation, but in many cases, simply making a new grid or preparing a fresh stain solution solves the issue. 

Microscopic black and white image of ngatie stain TEM stain crystal clusters  imaging | nanoparticle characterization at nanoimaging services
The negative stain TEM image of LNPs above shows an example of a dense uranyl formate stain cluster (pink arrows). The pink boxed region (left) is shown at higher magnification on the right. In this case, the stain clusters are few enough that they do not interfere with image interpretation. 

Cryo-TEM Grids Containing Carbon Film Artifacts

Cryo-TEM samples are often vitrified over a grid composed of a copper mesh that is covered with a carbon support film containing evenly spaced holes. These holes provide ideal imaging conditions because they contain only the vitrified sample. They have no added layer of substrate which would reduce the contrast between the particles and ice.

While particles of many sample types distribute evenly within the holes, some samples have a high affinity for carbon, resulting in a low number of particles within the holes available for imaging. These types of samples often benefit from the use of grids with a very thin carbon or graphene substrate covering the entire grid, including the holes. 

Grids that have a thin layer of continuous carbon covering the holes are made by coating a thin layer of smooth mica with ~4-5 nm of carbon. The layer of carbon is then floated onto water and transferred to holey carbon grids. During this process, small defects in the carbon layer can occur leading to artifacts in images. 

Vitrification of samples on grids with a thin carbon or graphene layer often helps to increase the number of particles within the holes. However, artifacts related to this thin substrate are sometimes visible within the image. The artifacts themselves vary in appearance, but are typically recognizable due to their relative high electron density.

Microscopic black and white image of cryo-TEM grids containing carbon artifacts imaging | nanoparticle characterization at nanoimaging services
The images above show an example of a carbon artifact on a grid with a thin continuous carbon layer that contains no sample. The purple boxed area is shown enlarged on the right.

If these artifacts are pervasive, unavoidable, and interfere with image interpretation or analysis, another grid is made. If needed, a new batch of grids are coated with carbon.

Drift Affecting Transmission Electron Microscopy Images

Although the time it takes to acquire a TEM image or movie is very short, if there is excessive movement of the sample the final image will appear blurred. Movement of particles can be induced by the electron beam or indirectly by breaking of the ice. A small amount of motion can be corrected for by alignment of individual image frames acquired on a direct detector.

Microscopic black and white image of cryo-TEM imaging | nanoparticle characterization at nanoimaging services
Movement of a lipid nanoparticle (LNP) sample shows up as a blurry image (left) and by the directional loss of Thon rings in the power spectrum of the image (right).
Microscopic black and white image of cryo-TEM imaging | nanoparticle characterization at nanoimaging services
After alignment of individual movie frames recorded on a direct electron detector, the image shown above is crisp and its Fourier transform has visible concentric circular Thon rings (right).

Less frequently, excessive movement is uncorrectable. When excessive drift is present in many images throughout data collection, the source needs to be identified. It may be that the grid is not secure within the cartridge or holder, the ice or grid substrate is too thin and unstable, or there are environmental vibrations that are affecting the microscope. Once the source of the drift has been identified and corrected, the sample can be reimaged.

Grid Imperfections

Grid imperfections resulting from a supplier’s manufacturing process can sometimes cause issues with image interpretation, but oftentimes only impacts the aesthetics of an image.

Microscopic black and white image of cryo-TEM carbon grid imperfections imaging | nanoparticle characterization at nanoimaging services
The images above are examples of imperfections in the carbon support film. Left: A minor amount of unevenness (blue curve) along the hole edge does not affect data quality. Right: A rough hole edge where the carbon support extends away from the edge (pink arrow) may affect image quality if it obscures a significant number of particles. In the above example it is minor and overlaps only one particle.

Overcome Cryo-TEM Imaging Challenges with NIS

While NIS encounters all of these imaging challenges and artifacts when collecting cryo-TEM or negative stain TEM images, our experienced microscopists have over 85 cumulative years of experience in preventing them to meet our clients’ need for quality TEM data. Our dedicated laboratory technicians and microscopists carefully optimize each grid in order to reduce image imperfections including crystalline ice, ice contaminants, stain crystals and pooling, carbon artifacts and more.

Contact us to talk to our expert scientists about how cryo-TEM or negative stain TEM imaging can assist in directly visualizing your nanoparticle formulations.

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