Top tips on sample preparation for image analysis

After the last Imaging Masterclass on good experimental practice I thought I would share my top tips for sample preparation, based on my own experience. You can view the recording of the webinar here.

Get to know your sample!

The first thing I would advise anyone to do is to look at their sample. Take a dry powder – by tumbling a pot of sample you can assess how cohesive or how sticky a sample is. Small particles, smaller than 10 microns or so can be very cohesive due to high surface energy and intramolecular forces. If you sprinkle some sample onto a micrscope slide this will tell you again how sticky the sample is based on how it disperses by hand. Now, use a micrscope (if using a Morphologi G3 you can use the manual microscope mode) and look at your sample. Do you see agglomerates? What particle size do you have? Is there a broad range of particle size? Do you have fragile looking particles, like needles?

Now you have some ideas about your sample you can make some decisions about dispersion.

Shall I disperse dry or wet?  

If the sample appears very cohesive then we may try wet. If there are many agglomerates and we wish to disperse those, perhaps wet will be better than dry. If particles have electrostatic charge then wet may also work best. A dry dispersion is of course is easier, so why not try a dry dispersion first and if results are not satisfactory then move to wet. (for more details on wet/evaporative dispersion see the Masterclass)

How much sample do I need?

If trying a dry dispersion with the Morphologi SDU here is some guidance on what sample volume to use based on particle size:

These are starting points for your dispersion but there is no golden rule as every sample is different.

What does a good dispersion look like?

When you have dispersed your sample take a look at the resultant dispersion. There are two example images of different dispersions below. The image on the left show a bad dispersion where too much sample has been used: too many particles are touching each other. The image on the right shows a good dispersion: there are still a couple of touching particles but also enough particles in the frame to make this an efficient dispersion to measure. This is what you want to aim for – as many particles as you can get per frame to reduce measurement time but minimise the number of touching particles.