Select samples correctly for biologically related knowledge. For mild microscopy earlier than vEM, repair with 4% paraformaldehyde, then use fluorescent probes or histological stains. For vEM, repair with phosphate-buffered glutaraldehyde and formaldehyde. Steadily dehydrate samples with growing ethanol, then clear them with an infiltration medium. Stain with heavy metals like osmium tetroxide and uranyl acetate for distinction. Then embed in appropriate brokers for sectioning. Paraffin embedding is reasonable, however epoxy resins are higher for ultrathin sections, though they want specialist tools and poisonous chemical compounds.

Quantity electron microscopy (vEM) is a department of electron microscopy (EM) that provides you biologically wealthy knowledge from more and more giant pattern volumes.

If you happen to haven’t already, try our introduction to vEM.

Are you performing or contemplating utilizing vEM in your analysis and on the lookout for recommendation on pattern preparation? Are you interested in the way it differs from pattern prep for different microscopy strategies?

In that case, to present you an thought of what to anticipate, we’ve assembled the principle issues that go into getting ready a pattern for a typical vEM experiment, together with an instance workflow and key concerns.

Select Your Pattern and Area of Curiosity Rigorously

There aren’t any two methods to slice it—vEM takes a very long time and offers you a heap of biologically advanced knowledge. So take nice care in deciding on an acceptable pattern, and guarantee it’s on the right developmental stage in your research.

Don’t simply go along with the primary pattern it’s important to hand as a result of it’s there—take the time to supply one thing beautiful by no matter metrics you may assess its high quality.

Pattern Preparation for Quantity Electron Microscopy: A Typical Workflow

There are some things value noting on the outset. The primary is that vEM will not be at all times a cryogenic technique like cryo-electron microscopy. You’ll typically chemically repair, stain, dehydrate, and embed your samples somewhat than picture them of their frozen hydrated state. Though word that there are cryogenic 3D imaging strategies reminiscent of cryo focussed ion beam scanning electron microscopy (FIB-SEM).

The second level is a consequence of this. Many pattern processing steps are irreversible, making your pattern incompatible with different imaging modalities. So, in order for you correlative knowledge or must picture one pattern in a number of methods, think about one of the best order.

1. Amassing and Fixing

Pattern preparation for quantity electron microscopy has a lot in frequent with different electron microscopy strategies.

First, you need to accumulate your pattern in line with its sort and repair it to cease it from degrading. Relying in your pattern sort, you would:

• Seed cells straight into glass-bottomed dishes reminiscent of MatTek dishes;
• Pellet your cell tradition;
• Harvest and part organs.

If you wish to do mild microscopy earlier than continuing to vEM:

1. Introduce fluorescent probes or stain your pattern with a dye that reveals what you need to see;
2. Repair it with 4% paraformaldehyde.

If you wish to go straight to vEM, repair your pattern in phosphate-buffered glutaraldehyde (2.5%) and formaldehyde (4%). This mix stabilizes the pattern’s construction by cross-linking proteins and nucleic acids whereas sustaining physiological pH. Permit the pattern to repair for so long as essential (1–2 hours, as much as a number of days, relying on the fixative, pattern sort, and pattern density).

Then, wash it with a phosphate buffer answer to take away extra fixative.

2. Dehydration and Clearing

Now it is advisable to take away all of the moisture out of your pattern. Dehydration protects your pattern within the vacuum chamber of the electron microscope for the reason that electron beam would evaporate residual water and hurt your pattern. Water additionally scatters and absorbs a portion of the electron beam, degrading the standard of your pictures.

Usually, you’ll dehydrate your mounted pattern by regularly exposing it to growing ethanol concentrations. For instance, 30–50%, 70%, 80%, and 90%. Every step ought to final ~15-Half-hour or longer if wanted. Then, end with an in a single day immersion of your pattern in absolute ethanol.

Gradual dehydration prevents distortion and shrinkage of delicate buildings in your pattern, sustaining its integrity.

And for one of the best outcomes, begin dehydrating your pattern instantly after you could have mounted it.

After dehydration, expose your pattern to an infiltration medium to take away residual ethanol. It’ll additionally make your pattern clear and extra porous (by eradicating lipids and different light-scattering compounds) so you may embed it in an acceptable medium and part it into skinny slices utilizing an ultramicrotome. This course of known as tissue clearing. For extra details about tissue clearing, join our tissue clearing masterclass. Frequent clearing brokers embrace:

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

No matter you select, guarantee it’s miscible (mixes with) along with your meant embedding medium.

3. Heavy Steel Staining

As talked about, vEM covers a spread of strategies, together with scanning electron microscopy (SEM) and transmission electron microscopy (TEM). For many samples, you’ll have to stain them with heavy metallic to supply distinction beneath the electron microscope.

Bear in mind, completely different heavy metallic compounds have completely different opacities to the electron supply inside the microscope, enabling you to differentially stain your pattern to (say) distinguish nuclei from mitochondria or proteins from nucleic acid.

Your staining protocol will rely in your pattern and what you need to see beneath the microscope. However typically, you’ll be working with options of:

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

These can be utilized to your pattern, probably in focus gradients, earlier than wicking away the surplus stain.

Try Desk 1 for a breakdown of what every stain preferentially binds to and offers distinction for.

Desk 1. A abstract of stains utilized in pattern preparation for quantity electron microscopy and the species they preferentially bind to.

Word that heavy metals are poisonous, so do your staining in a fume hood and put on PPE. And if staining fails otherwise you do it incorrectly, you’re going to get blurry pictures, so try this text for some basic EM pattern preparation ideas.

4. Embedding

Embedding allows you to part your pattern. 

Basically, you flip your pattern right into a compressed lump with uniform density, from which you’ll be able to take sections ≥ 50 nm thick by mixing it with an embedding agent and letting it set.

Keep in mind that for SEM, sections don’t should be as skinny as these for TEM, which might impression your alternative of embedding agent. 

There are a lot of brokers, together with paraffin wax, epoxy resins, and proprietary formulations. You’ll find a technical breakdown of those embedding brokers right here.

Paraffin is flexible, comparatively low cost, and first rate at preserving antigenicity. Massive air bubbles will be eliminated by gently tapping your pattern earlier than the paraffin units. As a result of paraffin infiltrates the pattern comparatively simply, smaller air bubbles normally rise to the floor as tiny voids are stuffed. 

Nonetheless, it may be laborious to supply ultrathin sections from paraffin-embedded samples. Plus, the embedded pattern is comparatively smooth, which means you may by chance harm it. For these causes, paraffin is extra generally utilized in fluorescence microscopy and immunohistochemistry. 

Epoxy resins polymerize to type a sturdy strong, defending your pattern after you could have embedded it. As a result of the polymer is strong, it’s simpler to supply ultrathin sections than paraffin. And it’s superior at preserving wonderful structural particulars inside your pattern. For these causes, it’s extra frequent in EM than paraffin embedding.

Nonetheless, embedding utilizing epoxy resins requires specialist tools since you are doing a polymerization response involving poisonous chemical compounds. Air bubbles are additionally extra of an issue, however you may take away these by rigorously bath-sonicating your pattern earlier than the medium polymerizes.

5. Sectioning and Imaging

One property of vEM that differs from many different EM strategies is that the sectioning and imaging steps are more and more built-in and automatic due to the big variety of sections wanted for 3D reconstructions.

This can be a slight simplification as a result of, in array tomography, all of the sectioning is finished earlier than imaging, whereas, in different approaches, sectioning and imaging are iterative. However your principal decisions are:

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

You’ll be able to examine their advantages and limitations in our introduction to vEM.

Array tomography is the most cost effective and most versatile, as it’s appropriate with TEM and SEM. It’s additionally the one non-destructive strategy.

Once more, it is a slight simplification since you are able to do FIB-TEM for those who like, nevertheless it isn’t typically employed in vEM, as a result of FIB- and SBF-SEM occupy the candy spot between part thickness, last decision, ease of automation, price, and so forth.

As an instance this level, you may purchase state-of-the-art SEM microscopes with an ultramicrotome in situ within the imaging chamber for totally automated SBF-SEM sectioning and imaging!

Useful Sources for vEM Pattern Preparation

It’s a wrap. Hopefully, this text has given you an thought of what goes into pattern preparation for quantity electron microscopy, the important thing selections you’ll have to make, and the professionals and cons of a few of the reagents and strategies obtainable.

vEM is an rising approach, and pattern prep will undoubtedly evolve because it enters the mainstream. For extra beneficial assets and protocols, try the vEM group webpage. Specifically, they’ve some sensible how-to movies that present you precisely easy methods to manipulate objects and samples when doing vEM.

ZEISS Microscopy additionally has a unbelievable vEM eBook you may obtain at no cost. It collates experiences from the vEM group so you may be taught from them.

To discover the superb knowledge vEM can present, try our webinar with Thermo Fisher Scientific: Showcasing the Energy of Quantity EM Throughout Disciplines.

Be taught extra about vEM and get knowledgeable perception into the way forward for electron microscopy strategies in The Microscopists Podcast episodes that includes Lucy Collinson and Kirk Czymme, and Kedar Narayan.

References

Bitesize Bio and Bitesize Bio-affiliated on-line assets had been used for reference and knowledge when creating this text, together with: