Transmission Electron Microscopy (TEM) is a high-resolution imaging method that supplies information regarding the morphology and structure of specimens. In order to penetrate samples, it utilizes a high energy electron beam and renders images using the transmitted part of the beam.
As highly accelerated electrons have a small wavelength, they can be employed to resolve small features. This capability is critical in nanotechnology, structural biology, and material science studies.
The sample to be imaged must be placed on a special metal grid, which is usually covered with ultra-thin (2-5 nm) polymer or carbon support. It has to undergo a glow discharge process first to ensure the surface support is suitable for use.
Figure 1. Native ferritin on in-air glow discharged carbon TEM support. Image Credit: Paul Simpson, Imperial College, London.
Why Do We Need to Glow Discharge TEM Grids?
As shown in figure 2, even freshly prepared carbon layers for TEM grids will have unwanted adsorbates such as water and low molecular weight material (LMWM) on the surface, usually adsorbed from the air. These contaminants must be removed using a glow discharge prior to utilizing the grids. This is done to ensure optimal sample adhesion.
Figure 2. A typical TEM grid with carbon film and representation of surface adsorbates. Image Credit: Quorum Technologies Ltd
The deposited carbon layer on the TEM grid has a variably charged surface, which is usually hydrophobic, so even spreading of the water-based sample suspension is extremely challenging, as shown in figure 3.
Figure 3. Wetting effect on hydrophilic and hydrophobic particles placed on hydrophilic and hydrophobic surface. Image Credit: Quorum Technologies Ltd
What is Glow Discharge?
A glow discharge plasma is a low-pressure gas that is partially ionized. It is made up of ions of net positive and negative charge. That quasi-neutral state is maintained by the presence of energetic electrons.
When inelastically colliding with gas molecules, these electrons ionize or excite them, leading to the formation of free radicals and ions of gas molecules. The characteristic glow which is seen during the process is due to photons that are released by neutralized free radicals (i.e., relaxation of electrons).
Glow discharge treatment is a known method that is utilized for cleaning and modifying surfaces. The result of the glow discharge treatment is dependent on the chosen plasma gas and polarity.
Figure 4. Glow discharge air plasma process. Image Credit: Quorum Technologies Ltd
Why are Different Types of Glow Discharge Needed?
Biological samples are the most difficult materials to prepare, as this process is made up of a number of equally important steps. Failure of one step can result in losing the sample completely.
The affinity of the molecule to the TEM supports might be different due to their specific local charge, which can be especially apparent in proteins, where specific parts of the molecule charge differently.
Adequately prepared TEM supports increase the number of retained molecules, enable the even spread of the sample and permit the molecules to be oriented on the TEM support to show their sides of interest. Staining the sample with heavy metals is also involved in the observation of biomolecules in ambient TEM conditions.
TEM supports that have not been treated with a glow discharge will lead to uneven staining, causing poor contrast in the image. This effect is also present in cryo-TEM, where the image can be unstable.
There are a number of reasons why making sure that the TEM grid is uniformly hydrophilic/hydrophobic and of the desired charge is crucial: the accurate placement of the sample on the grid, plunge freezing (in cryo-TEM), or staining and drying (in ambient TEM), and for imaging. The method of grid surface preparation and modification is dictated by the molecule to be imaged.
Figure 5. (a) Carbon support TEM grid before glow discharge with a droplet of water showing its hydrophobicity (b) Glow discharge treated TEM grid with a droplet of water showing its hydrophilicity (c), (d) Corresponding contact angle. Image Credit: Quorum Technologies Ltd
Selecting the Glow Discharge Method by Application
The desired effect of glow discharge is to make the surface of the TEM grid carbon support is appropriately modified and sufficiently charged for the application. A thin liquid film in which the sample is suspended will spread evenly and dry over the entire surface as a result of this. The below table shows examples of possible surface modifications together with their applications.
Source: Quorum Technologies Ltd
||No aggregation of particles on the grid square boundaries
||Hydrophilic (after subsequent treatment with magnesium acetate or 0.1% w/v polylisine)
||Better binding of nucleic acid to the grid surface
|TEM grids (for positively charged proteins)
||Covalent binding to the grid surface for positively charged molecules
|TEM grids (for proteins, antibodies and nucleic acids)
||Covalent binding to the grid surface negatively charged molecules
Advantages of a Two Chamber System
A compact, two-chamber system provides a fast, efficient way of trialing different types of surface modifications. This is ideal if the most common in-air glow discharge does not produce satisfactory results.
Image Credit: Quorum Technologies Ltd
A single chamber system is not as easy or reliable to use as a dual-chamber system when chemical vapor glow discharge is required. This is because of the chemical contaminants deposited during the process, which must be removed entirely by cleaning the chamber and system for in-air plasma treatment.
When using the chambers separately, the GloQube® Plus system prevents cross-contamination by using two chambers, and also offers no instrument downtime. A specially designed purge-pump cycle removes all of the remaining vapor chemicals used in the system.
The performance of the GloQube® Plus was exhibited in a series of cycles in methanol vapor tests. These were performed by utilizing a Hidden 200AMU RGA attached to the backing line, monitoring the residual gases. The tests exhibited no presence of contamination with CH3 O+ ion. The detailed results of the contamination test can be observed in the technical data.
Figure 1. Methanol(CH3OH) RGA cracking Pattern. Monitored gasses: CH3 at 15 amu, CH3O at 31 amu, CH3OH and O2 at 32 amu, CHO at 29 amu. Image Credit: Quorum Technologies Ltd
Figure 2. 1. Methanol introduced to 1x10-1 mbar and methanol capsule removed with bleed valve open. Recovery time 4 mins 2. Methanol introduced to 1x10-1 mbar for 3 mins and methanol removed with bleed valve open. Recovery time 4 mins 3. Methanol introduced to 1x10-1 mbar for 1 min and methanol removed with bleed valve closed. Recovery time 2 mins. Image Credit: Quorum Technologies Ltd
CH3 O is the most responsive gas to monitor, with a mass of 31 amu. Figure 2 demonstrates that if the methanol is removed from the input to the GloQube® Plus injection system, it takes approximately four minutes for the vapor to be pumped out of the needle and bleed valve assembly. It takes two minutes for the gases to be pumped out of the chamber when the bleed valve is closed.
Figure 3. A plasma was run for 30 mins in the vapor chamber with methanol. The chamber was vented and the door for both chambers was opened. The clean chamber was pumped down, and, when 1.5x10-1 mbar was achieved in the clean chamber, the RGA was switched on. Residual gases were monitored as the clean chamber continued to be pumped down. There is a slight decrease in all intensities at the start of this process, which is due to the vacuum level changing, and also due to the adjustment of the RGA operating pressure. No changes in the monitored peaks were observed after the initial calibration of pressure. There is no evidence of contamination from the vapor chamber to the clean chamber when methanol is used in the vapor chamber. It takes approximately two minutes for methanol to be removed, by pumping, from the vapor chamber after gas introduction. Image Credit: Quorum Technologies Ltd
Glow Discharge: In-Air Effect
A glow discharge air plasma is utilized for the hydrophilization and cleaning of surfaces. Oxygen from the air is ionized to negative and positive ions throughout the process, further reacting to form clusters.
These highly reactive species make the area extremely oxidized and uniformly negatively charged (in majority possessing carboxylic acid and ketone groups) by bombarding the surface and removing adsorbates (LMWMs). The process is non-destructive for the surface because of the low concentration of oxygen in the air, enabling its easy modification.
Most macro-molecules are hydrophilic, so they do not like to remain on a hydrophobic surface. This creates a requirement for the in-air glow discharge treatment of a carbon film support before their application on its surface, as shown in figures 6 and 7.
Figure 6. The effect of non-glow discharged carbon support TEM grid on retention, spread and staining quality of native ferritin sample solution. Low (6x10-4 μg/mL)and high(6x10-2 μg/mL) concentrations of the protein were used. Image Credit: Quorum Technologies Ltd
Figure 7. The effect of in-air glow discharged carbon support TEM grid on retention, spread and staining quality of native ferritin sample solutions. Low (6x10-4 μg/mL) and high (6x10-2 μg/mL) concentrations of the protein were used. Image Credit: Quorum Technologies Ltd
Native ferritin possesses hydrophilic 3-fold channels through which iron ions are transferred into the core. When utilized in low concentrations, it does not retain on a non-glow discharged carbon surface.
The formation of aggregates can be witnessed in high concentrations. Uneven charge on the carbon support also disturbs the proper staining, leading to light patches between protein groups. Changing the carbon surface using in-air glow discharge makes the surface negatively charged and hydrophilic, so the retention of native ferritin is considerably easier. In addition, negative staining of the sample with uranyl acetate works properly to give even staining, and the protein molecules can be observed correctly.
Methods of In-Vapor Glow Discharge and Advantages of Process Automation
Manual Gas Introduction
One of the first TEM sample preparation techniques utilized the manual introduction of vapor into a glow discharge chamber . Using this technique, a glass tube is filled with a few milliliters of chemical, and the flow into the glow discharge chamber is controlled by a manual Teflon valve. This deposits on the surface of the TEM grid once it is introduced and the glow discharge is ignited.
Placing a piece of cotton wool or filter paper saturated in the chemical into a glow discharge chamber is another well-known method of preparing TEM grids for sample dispersion. Once the chemical vapor is introduced, and the glow discharge ignited, the chemical is deposited on the surface of the TEM grid.
Using a capillary valve attached to an inlet nozzle on the chamber is another commonly utilized technique for chemical introduction into a glow discharge. The bottle of amylamine can then be attached to the capillary valve, and the flow of the vapor controlled manually. 
Figure 8. TEM grid carbon support modified by the blotting paper method and used for 20s proteasome sample application. Glow discharge system used: Quorum’s Emitech K100X. Disadvantage - the remains of oxygen and water vapor in the chamber disturbs the correct surface modification thus only a few side views appear on the grid. Image Credit: Quorum Technologies Ltd
Figure 9. GloQube® Plus with automated valve system, three consecutive runs showing the yield of side views of 20s human proteasome protein complex is over 80%. Image Credit: Quorum Technologies Ltd
Glow Discharge Using Chemical Vapor
The grafting of desired functional groups onto surfaces is enabled by introducing chemical vapor in the glow discharge process. This not only provides a method to change the wetting properties and charge of the surface, but it can also allow complex specimens to be bonded covalently to the grid’s surface.
For biomolecules like proteins and nucleic acids, this method of preparation is especially critical. In drug discovery and cancer research fields, understanding protein structure and their interactions with other macromolecules are of the utmost interest.
High-resolution electron microscopy must be utilized to gather the information needed for protein structure analysis and modeling. As revealing their active subunits is key for correct and successful data collection, the orientation of protein molecules on the TEM support grid plays a key part in this process.
Protein structure and spatial orientation usually need advanced methods to be used in order to direct their adsorption on the TEM support grid.
Glow Discharge Using Alkylamines
Alkylamines are known to form positively charged, hydrophobic films on carbon surfaces in glow discharge processes. These films negatively attract charged areas of the protein, enabling the observation of the desired orientation.
As the protein cores are usually also hydrophobic, their resulting hydrophobicity also helps to retain the molecules on the surface. The 20s proteasome protein complex is a known system for recognition and degradation of misfolded protein.
This is a well-established target for cancer therapy in an essential regulatory mechanism in cells. Amylamine (a homolog of alkylamine group) was employed to influence the orientation of 20s proteasome adsorption onto TEM grids.
Carbon support TEM grids were altered in a GloQube® Plus by utilizing an amylamine vapor glow discharge process to attain hydrophobic and positively charged surfaces to retain side-views of 20s proteasome complex.
Figure 10 shows the effect of altering the surface charge of the carbon support film on the orientation of the protein complex. Firstly, the freshly prepared carbon surface was non-uniformly charged and hydrophobic; so, a few of the side-views could be observed.
Figure 10. TEM images of 20s human proteasome complex showing the effect of altering the surface charge of the carbon support film on the orientation of the protein molecules. Carbon film of 2.5 nm thickness on Quantifoil 1.2/1.3 400 mesh was used as a support for the sample. Image Credit: Quorum Technologies Ltd
In the second instance, only top views could be seen after treating the grids with an in-air glow discharge, as this treatment makes carbon films negatively charged and hydrophilic. This process attracts the positively charged 19s part of the proteasome complex, leading to biased top-view orientation.
In the last instance, in amylamine vapor glow discharge, the majority of the 20s proteasome complex molecules were shown having side-view orientation.
Glow Discharge Using Alcohols
Alcohols, for example, methanol vapor, introduced into a glow discharge system will leave the carbon support grid slightly hydrophobic and negatively charged. A surface such as this will attract positive ions, such as native ferritin.
All ferritin molecules consist of 24 identical peptide subunits that fold into a spherical shell with a water-filled cavity inside. This cavity is connected to the outside via channels with threefold and fourfold symmetry and is thought to supply permeation pathways for iron ions and protons, vital for the proper functioning of ferritin as an iron depository.
Apo form of ferritin (an empty shell of ferritin) is also employed as an ion cage for the templated synthesis of nanoparticles – ZnSe or CdSe. Imaging of the load of the ferritin cage plays a key role in examining iron and other metals uptake.
An in-methanol vapor glow discharge treatment of carbon support TEM grids will help in the ferritin load study, and it will also stop the loss of ions through the channels. Figure 11 demonstrates that in-air glow discharge treatment of the TEM grid support enables the iron ions to be lost from the core, leading to empty ferritin (light areas inside the molecule core).
Figure 11. TEM images (Tencai F20 G2) of ferritin protein complex from horse spleen (Sigma Aldrich) applied to in-air and in-methanol vapor glow discharged carbon support TEM grids. Image Credit: Imperial College London.
Due to the negative charge and hydrophobicity of the surface, using in-methanol vapor treatment of the grid allows the user to keep the ions inside the core (dark load inside the ferritin molecules).
The GloQube® Plus supplies all the features necessary for TEM grid preparation when the glow discharge of these grids with air or vapor is needed. The utilization of a separate chamber for in-air and in-vapor glow discharge is essential to remove the risk of cross-contamination.
The GloQube® Plus design makes sure that no cross-contamination happens while also allowing the user to run processes sequentially using the same chemical without the requirement for cleaning in between.
As the flow rate can be controlled more accurately along with the chamber pressure, possessing an automated chemical injection system is also a huge benefit. This also supplies the ability to run longer process durations without the risk of exhausting the amylamine, as only a fixed amount is used - unlike the filter paper technique, for instance.
There is a continuous flow until the vial runs out with the automatic valve. A visual indication shows the level of chemical left in the vial. The added control provided by the automatic valve ensures repeatability and reproducibility of the TEM grid treatments.
The enclosed vapor delivery system with septum seal vial and automated valve system also minimizes operator exposure to the chemicals, increasing the safety of chemical use.
Image Credit: Quorum Technologies Ltd
 Dubochet, Jacques, et al. “A new preparation method for dark-field electron microscopy of biomacromolecules.” Journal of ultrastructure research 35.1-2 (1971): 147-167.
 Morris, E. P.; da Fonseca, P. C. A. High-Resolution Cryo-EM Proteasome Structures in Drug Development. Acta Crystallogr D Struct Biol 2017, 73 (Pt 6), 522–533.
 Aebi, Ueli, and Thomas D. Pollard. “A glow discharge unit to render electron microscope grids and other surfaces hydrophilic.” Journal of electron microscopy technique 7.1 (1987): 29-33.
Produced from materials originally authored by Dr. Anna E Walkiewicz from Quorum Technologies Ltd.
This information has been sourced, reviewed and adapted from materials provided by Quorum Technologies Ltd.
For more information on this source, please visit Quorum Technologies Ltd.