Desktop STEM as a sample screening tool for Cryo-EM

Sample preparation for cryogenic electron microscopy (cryo-EM) is a demanding and often expensive process, requiring several purification and screening steps to obtain a suitable specimen. Negative stain electron microscopy (EM) is one of the widely accepted sample preparation techniques for the initial screening of purified samples, and specifically for the assessment of pathology, morphology, concentration and agglomeration. Traditionally, a conventional transmission electron microscope (TEM) is used for this purpose. With the introduction of the desktop scanning transmission electron microscope (STEM), an alternative solution for screening samples has been developed that is much faster and more cost-effective than traditional methods.

Sample preparation for Cryo-EM

Cryogenic electron microscopy (cryo-EM) has become an invaluable tool in the field of structural biology, allowing researchers to visualize three-dimensional structures from 2D images at different orientations using advanced computational techniques at near-atomic resolution.

Cryo-EM is a labour-intensive and time-consuming technique that requires highly specialized skills. A crucial tool in sample preparation for (3D) TEM is the ultramicrotome.

A typical exponent of this technology, the RMC Boeckeler ATUMtome (Automated Tape Collecting Ultramicrotome) is capable of fully automatically producing hundreds to thousands of slices of several tens of nanometers thickness. This responds to the clear evolution from 2D to 3D (cryo)- EM. An interesting application note related to this is the imaging of mitochondrial protrusions in neuronal cells.

Read the Application Note here

Good Cryo-EM results also depend on the very high quality of the purified samples. Before a sample is vitrified for high-resolution imaging, it is usually screened to ensure that it is suitable for imaging and contains the expected dimensions and morphologies.

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Negative stain electron microscopy

In this technique, the sample is absorbed onto the grid and then a stain is applied to envelope it. A high mass-thickness contrast difference is produced by the background relative to the particles in the electron microscope, allowing for a “negative” image of the particles to be visualized at nanometer-scale resolution. The ease of grid preparation and high contrast provide an ideal method for assessing pathological details, morphology, concentration and agglomeration, among others.

Generally, a conventional TEM is used to visualize these samples. This leads to expensive equipment being used to screen samples instead of the intended application for high-resolution imaging. Facility costs and frequent maintenance costs can be significant, putting financial and time pressure on researchers who depend on the equipment for screening samples. Also, the learning curve for using a TEM can be quite steep: new users need days to weeks to become proficient in operating a TEM independently. Therefore, exploring alternatives to dedicated TEM instruments is an attractive choice for those who want to maximize the benefits of negative EM staining for sample screening.

Desktop STEM

In STEM-in-SEM, a SEM is used to produce transmission images. Recently, STEM-in-SEM has become available with the Thermo Scientific Phenom Pharos desktop SEM with STEM detector. This powerful combination of a desktop FEG-SEM and plug-and-play detector is compatible with standard EM grids and can capture bright field (BF), annular dark field (ADF) and high-angle annular dark field (HAADF) images.

Thanks to the Phenom Pharos resolution, which is better than 1 nanometer, obtaining STEM images (analogous to phase-contrast TEM images from traditional negative color EM workflows) takes just under two minutes once the sample is loaded. The compact size, intuitive software and hardware offer users significant savings in terms of facilities, maintenance and training costs. In addition, the Pharos SEM can be programmed to automatically collect data, allowing rapid inspection of the entire grid. This allows operators to collect a large number of images for screening in a short time.

Using the Phenom Desktop STEM for negative stain EM

Some examples of negatively stained biological samples imaged on the new Phenom Pharos Desktop STEM:

Above are some desktop STEM images of ultramicrotome kidney thin sections. These can be used for pre-screening, pathology and anatomical diagnosis.

Broken grid windows and well-stained regions

Contaminations and agglomerated samples

Above are some examples of how rapid pre-screening with SEM can be used to identify defects that occurred during sample preparation.

Virus-like particles stained with nano-tungsten showing rod-like and round morphologies. Based on these images, a microscopist can measure particle size and assess the purity, agglomeration and overall distribution of the sample to determine whether further optimization of the sample is needed for vitrification for cryo-EM.

Summary

For EM users looking for a cost-effective way to quickly perform EM work for pre-screening or diagnostic purposes, the Phenom Pharos Desktop STEM undoubtedly provides an excellent solution. The unit can be used to offload a high-res TEM, all at a fraction of the cost. In doing so, the Pharos STEM offers higher resolution and more contrast at lower KeV values than any other SEM.

Compared to classical TEM, the main benefits are:

Ability to image at lower KeV
Smaller investment and cost-of-ownership
Less training and experience required
Larger material range
Larger imaging range

With its versatility and affordability, the Phenom Pharos Desktop STEM is a valuable addition for any research facility looking to streamline its (cryo)-EM workflow.