A Simple Guide to Sample Preparation for Volume Electron Microscopy

Volume electron microscopy (vEM) is a branch of electron microscopy (EM) that gives you biologically rich data from increasingly large sample volumes.

If you haven’t already, check out our introduction to vEM.

Are you performing or considering using vEM in your research and looking for advice on sample preparation? Are you curious about how it differs from sample prep for other microscopy methods?

If so, to give you an idea of what to expect, we’ve assembled the main things that go into preparing a sample for a typical vEM experiment, including an example workflow and key considerations.

Choose Your Sample and Region of Interest Carefully

There are no two ways to slice it—vEM takes a long time and gives you a heap of biologically complex data. So take great care in selecting an appropriate sample, and ensure it is at the correct developmental stage for your study.

Don’t just go with the first sample you have to hand because it is there—take the time to produce something exquisite by whatever metrics you can assess its quality.

Sample Preparation for Volume Electron Microscopy: A Typical Workflow

There are a few things worth noting at the outset. The first is that vEM is not always a cryogenic method like cryo-electron microscopy. You will often chemically fix, stain, dehydrate, and embed your samples rather than image them in their frozen hydrated state. Although note that there are cryogenic 3D imaging methods such as cryo focussed ion beam scanning electron microscopy (FIB-SEM).

The second point is a consequence of this. Many sample processing steps are irreversible, making your sample incompatible with other imaging modalities. So, if you want correlative data or need to image one sample in multiple ways, consider the best order.

1. Collecting and Fixing

Sample preparation for volume electron microscopy has much in common with other electron microscopy techniques.

First, you must collect your sample according to its type and fix it to stop it from degrading. Depending on your sample type, you could:

• Seed cells directly into glass-bottomed dishes such as MatTek dishes;
• Pellet your cell culture;
• Harvest and section organs.

If you want to do light microscopy before proceeding to vEM:

1. Introduce fluorescent probes or stain your sample with a dye that reveals what you want to see;
2. Fix it with 4% paraformaldehyde.

If you want to go straight to vEM, fix your sample in phosphate-buffered glutaraldehyde (2.5%) and formaldehyde (4%). This combination stabilizes the sample’s structure by cross-linking proteins and nucleic acids while maintaining physiological pH. Allow the sample to fix for as long as necessary (1–2 hours, up to several days, depending on the fixative, sample type, and sample density).

Then, wash it with a phosphate buffer solution to remove excess fixative.

2. Heavy Metal Staining

As mentioned, vEM covers a range of techniques, including scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For most samples, you will have to stain them with heavy metal to provide contrast under the electron microscope.

Remember, different heavy metal compounds have different opacities to the electron source within the microscope, enabling you to differentially stain your sample to (say) distinguish nuclei from mitochondria or proteins from nucleic acid.

Your staining protocol will depend on your sample and what you want to see under the microscope. But generally, you’ll be working with solutions of:

• Osmium tetroxide;
• Potassium ferricyanide;
• Uranyl acetate;
• Lead citrate;
• Lead aspartate;
• Ruthenium red.

These will be applied to your sample, potentially in concentration gradients, before wicking away the excess stain.

Check out Table 1 for a breakdown of what each stain preferentially binds to and provides contrast for.

Table 1. A summary of stains used in sample preparation for volume electron microscopy and the species they preferentially bind to.

Note that heavy metals are toxic, so do your staining in a fume hood and wear PPE. And if staining fails or you do it incorrectly, you will get blurry images, so check out this article for some general EM sample preparation tips.

3. Dehydration and Clearing

Now you need to remove all the moisture from your sample. Dehydration protects your sample in the vacuum chamber of the electron microscope since the electron beam would evaporate residual water and harm your sample. Water also scatters and absorbs a portion of the electron beam, degrading the quality of your images.

Typically, you will dehydrate your fixed sample by gradually exposing it to increasing ethanol concentrations. For example, 30–50%, 70%, 80%, and 90%. Each step should last ~15-30 minutes or longer if needed. Then, finish with an overnight immersion of your sample in absolute ethanol.

Gradual dehydration prevents distortion and shrinkage of delicate structures in your sample, maintaining its integrity.

And for the best results, start dehydrating your sample immediately after you have fixed it.

After dehydration, tissue clearing makes your sample transparent and more porous (by removing lipids and other light-scattering compounds) so you can embed it in a suitable medium for sectioning into thin slices. For more information about tissue clearing, sign up for our tissue clearing masterclass. Common clearing agents include:

• Propylene oxide;
• Acetone;
• Xylene;
• Toluene.

Whatever you choose, ensure it is miscible (mixes with) with your intended embedding medium.

4. Embedding

Embedding your sample by infusing it with an embedding agent and letting it polymerize or set enables you to take sections from it.

Remember that for SEM, sections do not need to be as thin as those for TEM, which could impact your choice of embedding agent. 

Epoxy resins polymerize to form a durable solid, mechanically protecting your sample after you have embedded it. Because the polymer is solid, it is easier to produce ultrathin sections from than soft embedding agents.

However, embedding using epoxy resins requires specialist equipment because you are doing a polymerization reaction involving toxic chemicals. Air bubbles are also more of a problem, but you can remove these by carefully bath-sonicating your sample before the medium polymerizes.

5. Sectioning and Imaging

One property of vEM that differs from many other EM methods is that the sectioning and imaging steps are increasingly integrated and automated because of the large number of sections needed for 3D reconstructions.

This is a slight simplification because, in array tomography, all the sectioning is done before imaging, whereas, in other approaches, sectioning and imaging are iterative. But your main choices are:

• Array tomography;
• Serial block-face imaging (SBF);
• Focussed ion-beam milling (FIB).

You can read about their benefits and limitations in our introduction to vEM.

Array tomography is the cheapest and arguably the most versatile, as it is compatible with TEM and SEM so long as sections suitable for the relevant application are taken separately. It’s also the only non-destructive approach of the three listed.

Again, this is a slight simplification since you can do FIB-TEM if you like, but it isn’t often employed in vEM, because FIB- and SBF-SEM occupy the sweet spot between section thickness, final resolution, ease of automation, and cost at the time of writing.

To illustrate this point, you can buy state-of-the-art SEM microscopes with an ultramicrotome in situ in the imaging chamber for fully automated SBF-SEM sectioning and imaging.

Helpful Resources for vEM Sample Preparation

It’s a wrap. Hopefully, this article has given you an idea of what goes into sample preparation for volume electron microscopy, the key decisions you will have to make, and the pros and cons of some of the reagents and methods available.

vEM is an emerging technique, and sample prep will undoubtedly evolve as it enters the mainstream. For more valuable resources and protocols, check out the vEM community webpage. In particular, they have some brilliant how-to videos that show you exactly how to manipulate objects and samples when doing vEM.

ZEISS Microscopy also has a fantastic vEM eBook you can download for free. It collates experiences from the vEM community so you can learn from them.

To explore the amazing data vEM can provide, check out our webinar with Thermo Fisher Scientific: Showcasing the Power of Volume EM Across Disciplines.

Learn more about vEM and get expert insight into the future of electron microscopy techniques in The Microscopists Podcast episodes featuring Lucy Collinson and Kirk Czymme, and Kedar Narayan.

References

Bitesize Bio and Bitesize Bio-affiliated online resources were used for reference and information when creating this article, including:

• Showcasing the Power of Volume EM Across Disciplines. Webinar with Jurgen Kriel. Sponsored by Thermo Fisher Scientific.
• The Microscopists episode #62. Podcast with Lucy Collinson and Kirk Czymmek. Sponsored by ZEISS microscopy.

Thomas Warwick

I have a Master’s Degree in Chemistry and a Ph.D. in Structural Biology. I am interested in how the shape and connectivity of molecules relate to their reactivity and function.

Share this article: