Atomic force microscopy (AFM) can be really tricky – understanding how it works, making sure you choose the correct AFM probe, optimising parameters... On top of this you do not want to make life harder for yourself with poor sample preparation.

Below I will share a few tips I have picked up from the various labs I have been fortunate to work in.

I will do my best to give you advice on how to prep good samples, but it is often trial and error that leads to the best sample prep. Once you have read all the advice, be sure to get stuck in and adapt as you go.

Jamie’s Top Tips

1. Choose the right substrate, and make sure it is flat enough!

There are a variety of things to consider when choosing a substrate for your AFM sample. The most important consideration is the roughness. If you are looking at samples that have step heights of 100s nm then it will not matter too much, but if you are looking at samples with step heights of only a few nanometres (such as biomolecules) then it is essential to use an atomically flat surface.

Each type of substrate has pros and cons. Common examples include:

Mica - an atomically flat surface that is used regularly to adsorb biological samples on to. It is easy to prepare and single layers can be cleaved away using tape to reveal a new clean atomically flat layer.
Glass - transparent so is useful for combined AFM with optical microscopy. Glass is rougher than mica, which can affect AFM results, particularly if your sample has small height steps.
Silicon/Silica - silica has a similar surface chemistry to glass but is much smoother.
HOPG (highly oriented pyrolytic graphite) - atomically flat and used for conductive measurements.
Gold – useful for surface modifications such as attachment of polymer monolayers or attachment of bio-molecules via thiol links. Can be atomically flat if deposited correctly. Not easy to buy so you may need the expertise to thermally deposit gold layers in-house (for reference - mica, glass, silicon and HOPG can be bought quite easily).

2. Stock up on these essentials!

Once you have chosen a suitable substrate, you will likely need a way to attach it to the AFM stage. This is when you will need these essential items below.

Magnetic Metal Stubs

Agar Scientific AFM Metal Specimen Discs

These are incredibly useful for attaching flat substrates to, so they can be put on the AFM sample stage. Most types of commercial AFM have magnetic stages.

Much like loose change and pens, they have a habit of going missing or rolling under desks, so it’s best to have a good stock of them. You can even re-use them by cleaning off the sample and any glue/tape with solvent and scrubbing.

Double sided tape

Double sided tape is your friend for fast sample prep. Regular tape from the local shop is fine, or you can use more specialist adhesive carbon tape. However, be careful if you require low noise and high resolution of small structures, as there can be lateral and vertical sample drift with tape. Tape is particularly helpful if you need to re-use the sample for another instrument or measurement.

Glue

Glue is great if you want to minimise sample drift, but can limit the re-use of the sample afterwards. I recommend two types of useful glue:

Epoxy glue is easy to get hold of (you can buy it online or in a hardware shop). It normally consists of two precursor gels that you mix, and the glue sets in under 5 minutes.

UV cure glue can set in seconds when activated by UV light. With epoxy glue, even though you have a few minutes for it to fully set, it starts to set as soon as you mix it, so you are acting against time. If you make a mistake or mis-position the sample, it is hard to correct! With UV cure, you can reposition the sample as many times as you like and activate the glue once the position is perfect.

You will need to make sure you have a UV torch or lamp with the correct wavelength for the glue you have. Make sure to take the necessary safety procedures (mainly not shining in your eyes).

UV cure glue will only work for transparent substrates i.e. mica or glass. You cannot shine UV light through opaque substrates!

Substrates

It is also good for any AFM lab to be stocked up with the substrates from tip 1, especially the more commonly used and cheaper ones like mica, glass slides and silica.

3. Be clean!

The resolution of an AFM is amazing – one angstrom in the vertical Z direction and as low as one nanometre in the lateral XY direction. This is great for your high-resolution imaging of cool samples but will also show up contaminants in high-resolution! You will have never seen a speck of dust in such high resolution!

Contaminants that are small by eye, can be huge in an AFM image and even take up the whole image. This will result in you struggling to see your sample and having to check many areas which is time-consuming, and may change your sample structure, interactions, and chemistry.

It’s best to keep everything clean! Store your samples correctly (e.g. fridge, freezer or under vacuum), use gloves, and make sure you know an appropriate way to clean your samples without destroying or degrading them.

4. For biological AFM, don’t let buffer dry out!

Imaging in liquid opens a whole world of biological imaging in close-to-physiological conditions.

However, you must make sure you keep your sample hydrated. If the water evaporates:

The laser may misalign on the cantilever, and you will not be able to image.
The nicely assembled bio-molecules or cells that rely on water for their structure will be destroyed if the water completely dries out.
The concentration of salts in your finely balanced physiological buffer will change as water evaporates. If you lose half the volume of water, the salt concentration will double. This could have a massive effect on cells, DNA, or proteins.

There are two main ways to solve this issue:

The easiest is to monitor the water level by eye and top it up with a pipette. This only works if you have an open liquid cell.
The second is to use a closed liquid cell which can stop evaporation (many AFM manufacturers offer these). It will also have an input for controllably adding/changing buffer.

If you have evaporation and want to keep your buffer concentration consistent, be sure to add in pure water, not more buffer. Otherwise, you will be artificially increasing the salt concentration.

5. Check the literature

If you have a new sample you want to prepare for AFM, it can be hard to know what is best. Always check publications because there may be researchers who have already tackled similar materials with AFM imaging. Check for information in the method sections which you can adapt.

In fact, it is not just AFM papers that might be useful. Sample preparation on flat surfaces is required for other surface sensitive techniques e.g. optical microscopy, x-ray diffraction, and electron microscopy. You may find a method that can be adapted for AFM. In particular, many other techniques use glass substrates.

Finally, let us know if you have any good AFM sample prep tips and tricks. This blog is certainly not exhaustive. I know I learn new tips all the time, both from my own experience and from colleagues and customers. When you work in AFM, you never stop learning, and we love to be informed by the community and our customers.

If you enjoyed this blog post, you might also like 5 top tips for effective AFM imaging: a beginners guide or 5 more top tips for effective AFM imaging: the intermediate guide.