High-Resolution Episcopic Microscopy (HREM) Protocol

The difference between ethanol and methanol in sample dehydration is that methanol can be 100% pure, while ethanol usually contains a small amount of water. If your sample needs to be completely free of water, such as using Technovit 8000 (which is moisture sensitive) methanol is better. However, ethanol can be used as an alternative with JB-4.

• Don’t embed the sample right away—wait until the JB-4 has started to set slightly. It should still be a bit runny but firm enough to hold the sample in place.

• Use a torch (a phone light works) to shine through the PTFE mould so you can check the sample’s position.

• To adjust the orientation, use flat capillary tubes with the ends gently heated and rounded to avoid damage.

Try to keep handling to a minimum to reduce the chance of introducing air bubbles.

If your blocks come out of the mould feeling very soft, rubbery, or jelly-like, there may be an issue with the JB4 mix or one of its components. A slight springiness is normal, especially in larger blocks, but they should still feel firm overall. If needed, you can harden them by baking at 90–100 °C for a few hours or overnight.

Even fully cured blocks can soften slightly over time, whether stored at room temperature or in the fridge. The exact cause isn’t clear, but this tends to happen more often during warmer months in the UK. Re-baking usually restores the hardness.

This can happen if not enough JB4 has set through the central hole and crosshair gaps in the base of the chuck.

To prevent this, add a small amount of fresh polymerisation mix to fully fill the base.

If the block has already broken off, use abrasive paper to flatten both the surface of a new chuck base (they’re often uneven from manufacturing) and the bottom of the JB4 block.

Then reattach the block using SuperGlue or a similar contact adhesive. We have found that additive resin glues such as araldite also work well (if not better).

This usually means the sample wasn't infiltrated long enough. Infiltration time is important not just for letting the methacrylate fully penetrate the tissue, but also for allowing the dyes to stain it properly. For reasons we don’t completely understand, the staining seems to take longer and is key to getting good image contrast. Dense tissue is especially prone to showing a bright, featureless centre if infiltration is too short. To fix this, try extending the infiltration time. Just keep in mind that longer infiltration can also darken the overall grayscale in your images.

These wavy lines, usually running along the direction of the cutting blade, are often caused by JB4 that hasn’t fully hardened. This is more common near the base of the block, where polymerisation can be weaker—likely due to oxygen exposure interfering with the setting process.

Typically, JB4/dye mix taken directly from the fridge should start polymerising within 15 to 30 minutes, becoming viscous and warming up if there's any surplus. If it's taking much longer or not polymerising fully, check your components by testing JB4 without dyes. Incomplete removal of methanol during sample infiltration can inhibit polymerisation.

Yes. You can trim them by briefly baking the block at 90–100 °C for 5 to 10 minutes. This softens the resin, making it flexible for about 1–2 minutes after coming out of the oven. During that time, you can cut it using a single-edge razor blade or utility knife with a steady push and gentle sawing motion. Work quickly—once the block cools, it hardens again and becomes difficult to cut.

After adding the catalyser, shake or vortex the mixture, then let it sit for about a minute if you're using around 20 ml—longer if you're working with a larger volume. Then sonicate the mixture, you’ll notice bubbles rising and popping.