Hi,
We are imaging self-assembled nanoparticles on optical fibers. Our surface is highly charged, and tends to attract a lot of junk. We've eliminated a lot of contamination believe it or not, but occasionally still get surprised in our pictures. I was hoping to show off some of these images and see if anyone can identify the likely source of the junk we're seeing:
First, there's this rope-like material that I would best is microbial.
The second one kind of happens randomly- it seems like there are coatings/pockets of maybe sticky stuff over our monolayers. We notice it seemingly at random; however, it usually conincieds with experiments involving proteins; however, most of our experiments where we subject a fiber to proteins do not have this. Maybe such a contamination is already characterized somewhere?
We are taking as many precautions as possible; storing fibers in a vacuum-umpped box; filtering all solutions through millipore buffers etc..., but our samples sometimes go a week, in and our of the fridge, in and out of solutions, and eventually we see something appear on the images.
The contaminants I'm showing usually appear in local areas on the surface rather than completely coating the entire surface.
bacterial cell division could be used to differentiate a bacteria and an artifact if it is spherical bacteria. the smallest bacteria ureaplasma could be as small as 300/400nm. but in the top picture, as the spheres are smaller than 100nm, possibly these are vesicles / sort of exosome like structures. in the bottom picture, could be microbial but possibly not bacterial owing to the size of the rope like material.
while loading the sample before coating by sputter..i think you do not functionalise the slides on which the sample is loaded. you can use paraformaldehyde or ethanol to funcationalize the slides and after you are done . load your sample on those slides. and then dry them under uv lamp. then coat it .. these are not bacteria. at this resolution you get a pretty large size of bacteria i have worked on the anti-biofilm activity of nanoparticles on bacteria and i think these are not bacteria. please provide me the magnification used. and the probe current as well as voltage values ?
OOPS!
Sorry, the spheres Sriram are gold nanoparticles. These are supposed to be there.
Uzair, first let me say we aren't sputter-coating at all. The gold is a self-assembled monolayer, used in an experiment, then washed in filtered water (sometimes ethanol, but I think water is more fair since I don't want to rinse anything off that might have been present during the experiment).
The top pic is 50kx, the bottom is closer to 5k or less. It has a scale bar on the bottom if that helps. The operating voltage is 1KeV only. The AuNPs are 22nm diameter. If the magniciation is paramount, I can go dig it up.
There are no bacteria on your specimens.
I have seen a lot of contamination sites similar to your second picture. Usually it is some mechanical transfer of organic stuff on a specimen (often on edges of specimen). Contamination on the first picture definitely come from solution, it looks like you have some crystals (grayish sticks). Do your solutions contain salts?
I would hazard that both types of contamination are organic. Perhaps just unstructured carbon gel or biofilm in the first image. If you increase the Kv does it dissappear or become more ghost like? Have you compared with EDS areas with and without contamination? Does the carbon peak change in counts? The second image looks almost fungal; i.e. hypha that have been distorted by the vacuum.
Hi Vlad. The crystals are probably saline; we use phostphate buffer saline and have verified that if we don't rinse the fiber in water, the surface is covered in salt. On this particular sample, maybe we didn't let it rinse long enough.
Marjorie, We haven't tried increased Kv, but we can looka t it. No, we haven't done any EDS. We can try it, thanks.
I've posted one more image from a related sample. This surface was mostly clean except for a few localized bubbles. All beam settings are identical. This was very strange because we never saw such highly-contrasted bubbles before. Just thought I'd share because you guys seems to know a lot about these images.
Adam, That last almost image looks like a liquid with high surface tension. Could the vacuum have "dried" the contamination into these blebs? Are the vacuum conditions constant? For example, are some samples evacuated more quickly than others? Grasping at straws, but might help you consider what may be different about the contaminated samples. I once keep a sample in a desiccating jar and spent an hour or so imaging this cool stuff on the SEM only to find out the Ba containing sediment was the desiccant.
The last image actually was rinsed in ethanol rather than water prior to imaging. Maybe the ethanol could have tension? Otherwise, the vacuum conditions are constant.
Yes, that explanation does make sense, but I guess without a secondary analysis method like EDS as was previously mentioned, I can only guess at what I'm seeing. Probably could have dried the contamination though, never thought about that!
Without doubt it is dried contamination, but I am not sure it was washing solution (ethanol) that left these blobs. Ethanol does have notable surface tension and about the same viscosity as water. However usually it has good wetting ability and so it is difficult to explain large contact angle of the blobs. To form contamination particles like these drying solution (ethanol + some organic stuff) should have not too high adhesive forces between liquid and substrate. May be it can help to find source of contamination.
Another possibility – some liquid was sprayed on already washed and dried specimen. Mist from a pipette? Somebody sneezed?
Adam,
From my own SEM experience, I've leaned of never going fishing with SEM! Still, perhaps the fingerprints of these objects may be so unique that somebody would have already completed all required steps (beyond SEM) for their identification and could share it with you.
Clean the sample and SEM chamber with remote plasma cleaner (www.piescientific.com) may be able to help you to achieve better results. SEMI-KLEEN plasma cleaner generates radical atomic oxygen and ozone that can remove organic contaminants from sample and chamber.
http://www.piescientific.com/Product_Plasma_Cleaner
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