Is your sample preparation holding you back? by Chris Parmenter
So often we obsess over what resolution we can obtain and as instrument design advances and limits are driven down, we expect that the data and clarity of images from our instruments will naturally improve. Perhaps your microscope is new, or maybe it’s older, either way if imaging is proving tricky it is easy to blame the instrument itself. Certainly the age or specification is a factor (field-emission instrument or not), as can be particular issues with a sample - like conductivity, but what I’m talking about is preparing your samples in the best way to give the data you actually want and need. Maybe it is time to ask, “How good is your sample prep?” I’ve talked about this with many people and they all say the same thing, “garbage in, garbage out” - basically our samples have to be prepared with as few artifacts and defects as possible, to take advantage of the field-emission and aberration-corrected instruments that are becoming commonplace.
Take the example of a polished sample that is first cut with a blade or saw, embedded and then ground and polished. Assuming the final paste used 1 µm diameter abrasive particles, then we can assume there will be marks on the surface about 3x the diameter of the polishing particles. With potential striations 3 µm in diameter it will be difficult to see sub-micrometer features, let alone the 50 nm or smaller nano-scale features you were after! And what if your sample picked up some contamination whilst being stored or transported? Would you know? I’ll be honest, I am pretty trusting of samples when I receive them, but recently I was startled to see some images of samples before and after cleaning using a UV cleaner such as the ZoneSem cleaner, which made me think about sample prep in general. Two approaches in particular caught my eye. The first was UV cleaning, which is gentler than plasma cleaning and, as the images below show, the invisible contamination has a drastic effect on the observable features.
Images of tin on carbon, sputtered with gold. The sample was left inside an oil rotary pumped sputter coater over night. Left: No fine surface details visible. SEM scanned area shows strong contamination build-up. Right: Same sample, cleaned for 25 minutes in the UV cleaner. The hydrocarbon film has been effectively removed, the fine Au grains are clearly visible without new contamination build-up within the scanning area. (Images and text courtesy of Hitachi High Technologies)
Once the sample is clean, here’s a thought, what about your sample chamber? Maybe that needs to be cleaned in order to keep your now pristine samples clean. As we move to SEM and FIB-SEM instruments being used in multidisciplinary settings and increasingly with biological samples, there is a greater likelihood that the chamber becomes contaminated. To counter this issue, manufacturers of such instruments are now offering ‘in chamber’ plasma cleaners as a standard add-on, like the Evactron by XEI. By regular cleaning of the chamber, the column and apertures can be maintained to ensure the microscope is at its peak resolution.
So far I’ve talked mostly about SEM issues, but what about TEM? In mid-September, I helped to organise an ultramicrotomy course with RMC and Cambridge Nanoscience, where we learned that most materials can be sectioned using a microtome, if the preparation is right. That means that if the sample is cut at the correct temperature (room or below zero, down to -120oC), then the result is properly sectioned samples, that can be transferred to the TEM grid and imaged. Of course prior to this step, the sample must be prepared by embedding or stabilising the samples in some way and that in itself requires the proper approach and knowledge of the embedding medium or the protocols required to fix the structures and replace the water with resin and stain.
The overall message is: Get the right sample preparation, whether it be microtomy, grind and polish, focused ion beam, high-pressure freezing or freeze-substitution. With the right approach, it can really make the difference and allow you to observe what you want to see or measure. Failure to do this can subtract from your available resolution or fail to preserve the objects of interest.
What is your view of sample preparation, do you have an opinion you would like to share on this topic? If so, please let us know.
Dr Chris Parmenter