Transmission Electron Microscopy (TEM)

Vironova revolutionizes transmission electron microscopy (TEM) for biopharmaceutical development based on image analysis. TEM is a microscopy technique in which a beam of electrons is transmitted through a sample to form an image at a higher resolution than light microscopy, such that fine details can be captured. TEM can be an extremely useful analytical tool for characterizing, for instance, viral vectors and drug delivery systems.

At Vironova, we offer a complete GMP-certified TEM analysis package that includes both negative stain (nsTEM) and cryogenic TEM (cryoTEM) analyses for biopharmaceutical products.

GMP-certified electron microscopy laboratory

Good Manufacturing Practice (GMP) is the part of quality management concerned with production and quality control, which ensures that products are consistently produced and controlled to the quality standards appropriate to their intended use and as required by the product specification.

Vironova has the world’s first GMP-certified electron microscopy laboratory providing TEM services. All of Vironova’s EM Services personnel are well-trained and qualified. The Vironova Analyzer Software (VAS), the only GMP-compliant TEM analysis software in the world, can help organizations to be FDA 21 CFR Part 11 compliant. VAS enables the extraction of valuable information through image acquisition, analysis, and reporting, while providing accuracy and traceability.

Vironova’s premises and equipment are well-established, validated, calibrated, and maintained according to GMP guidelines. In our GMP-certified laboratory, we can conduct work on samples that are classified as biosafety level 1 and 2 (BSL1 and BSL2). The BSL assigned to a client’s Product and project is based on a biological risk assessment performed by our laboratory safety team. Any deviations and changes in the laboratory work are handled under quality control according to our standard operating procedures.

In our GMP-certified laboratory, we also provide our clients with a validation of the Method of Analysis (MoA). The purpose of the method validation for a client’s specific product is to ensure that test results generated from a specific test procedure, i.e. nsTEM or cryoTEM analysis, and for a specific Product or range of Products are reproducible, accurate, and reliable within an acceptance criterion.

Table 1. GMP-certified and non-GMP analyses

In general, TEM analysis involves nsTEM where the sample is prepared and imaged at room temperature, as well as cryoTEM, where the sample is prepared and imaged at cryogenic temperature (around -180°C). The difference in sample preparation and imaging operation are described below.

nsTEM (room temperature preparation)

For nsTEM, the grid preparation procedure involves depositing a sample onto a TEM grid and staining it with a heavy metal salt solution. The staining solution acts as a sample embedding agent and as a contrast agent, as it is composed of electron dense atoms that diffract electrons in the TEM beam. The nsTEM technique allows for room temperature investigation of viral vectors: from sample preparation to imaging within the microscope. In some cases, the sample can be subjected to dehydration effects or undesirable interactions with the staining solution. However, the image contrast achieved using nsTEM is better than with cryoTEM, and particle surfaces and backgrounds can be more easily distinguished.

Figure 1. Schematic of the nsTEM sample preparation

nsTEM details

• Glow discharge the TEM grid
• Apply the sample (3 µL) for 60 seconds
• Blot off the excess sample with filter paper
• Wash the grid with distilled water (3 µL)
• Blot off the excess water with filter paper
• Apply stain solution (3 µL) for 10 seconds
• Blot off the excess stain solution with filter paper
• Dry the grid at room temperature for ca. 10 min

Tutorial
Tutorial for this protocol 

Figure 2. Example nsTEM images of various particles

cryoTEM (cryogenic preparation)

For cryoTEM, the grid preparation procedure involves depositing a sample onto a TEM grid and plunge-freezing the sample in a cryogenic liquid, such as liquid ethane, at temperatures around -180 °C. The sample is embedded in an amorphous water ice (i.e. non-crystalline ice), preserving the sample in a hydrated and close-to-native state. The prepared grid is then stored in liquid nitrogen until its insertion into the microscope, and it is imaged under cryogenic conditions.

Figure 3. Schematic of the cryoTEM sample preparation

cryoTEM details

• Glow discharge the TEM grid
• Apply the sample (3 µL) within a humidifier chamber, at 99 % relative humidity and 16 °C, and incubate for 10 seconds
• Blot off the excess sample with filter paper
• Plunge the grid in liquid ethane (-184 °C)
• Store the grids in liquid nitrogen (-178 °C) until transfer to the electron microscope for imaging

Figure 4. Example cryoTEM images of various particles

What can be analyzed with TEM?

At Vironova, the main application areas we offer expertise on are within nanoparticle characterization in the fields of gene therapy, vaccines, and drug delivery. Direct imaging of viral vectors, for instance, is extremely useful for distinguishing the morphological features within a sample to answer not just one, but several critical questions, such as:

• Have all the host cell debris been removed during the downstream purification process?
• Has the particle morphology or integrity degraded due to unsuitable downstream conditions?
• Have product aggregates formed during upstream operations or due to unsuitable downstream conditions (e.g., purification-inefficient packing causes empty capsid impurities)?
• How many different types of morphological particles are present in the sample, and what is the particle percentage of each domain?

Figure 5. Manufacturing process of viral vectors

Which TEM technique to choose?

When you are deciding on the most suitable TEM technique, nsTEM or cryoTEM, for analyzing your sample, it is important to understand the different characteristics of each technique and the morphological features that can be resolved. The following table shows the differences observed when using nsTEM and cryoTEM for imaging a sample.

Table 2. Different views of the sample in nsTEM and cryoTEM

In nsTEM, the sample is embedded in and stained with a heavy metal salt solution, which enhances the contrast level during imaging. This allows for good visualization of the surface structures of the particles within a sample, as well as background details. This technique is particularly suited for investigating particle overall morphology, size distribution, and purity. Additionally, it can be used for assessing particle integrity. Due to penetration of the staining solution into a broken particle, its interior has a dark and inhomogeneous appearance surrounded by a well-defined exterior, while an intact particle’s interior appears bright and homogenous. To lend insight into sample aggregation, particle clustering/aggregation can be analyzed by detecting both individual and clustered/aggregated particles and classifying them accordingly, whereby the percentage of each class (individual or clustered/aggregated particles) can be revealed. Additionally, size or area distribution of clustered/aggregated particles can also be determined.

In cryoTEM, particles are fixed in vitreous water ice and imaged under cryogenic conditions. This allows the sample to remain in a hydrated or close-to-native state. Therefore, the most outstanding characteristic of the cryoTEM technique is that it is possible to resolve the internal structure of the particle. For instance, cryoTEM can differentiate the filled, empty, and intermediate AAV particles according to their pixel intensities.

Table 3. Summary of nsTEM and cryoTEM analyses