If I was asked to do something like your task, I would avoid any specimen preparation (at least of first stages) and will work without additional conductive coating. Blood plasma could be identified by presence of crystals of chlorides (mostly NaCl) and by thin layer of proteins. Islands of protein “coating” should be visible as darker areas on BSE pictures (go as low in voltage as your BSE detector allows). Bigger coagulated plasma clots can have fibrous structure, but it is less probable and need proper specimen prep (fixation, dehydration..) You may also try (micro)Raman spectroscopy.

I am not sure if SEM images ia good method to see biomolecules maybe if you obtained a mapping picture before and after treat the electrodes with big blood plasma you can see any difference in the Au distribution.

I would go by using AFM - a less invasive approach. Normal tapping mode at 1 Hz scan rate on a 10 micron scan size at 512 pixel resolution should resolve a lot of questions. I would SEM image the AFM tip before and after the AFM scans just to be on the safe side before drawing any conclusions.

Thanks a lot Vladimir, by the way can you suggest me the method for the SEM specimen preparation (fixation, dehydration).

Krishna Mohan: Thank you very much for your suggestion. I will try AFM also.

Logudurai, if you really want to see biomolecules (as I understand proteins of a blood plasma) as single entities, then SEM is definitely wrong instrument for you. May be you can use immunostaining (not sure) for optical scope; immunogold staining for SEM will not work for you – gold on gold will be invisible.

Actually I think it would be possible to identify immunolabeled proteins. Colloidal gold, due to its round shape should be distinguishable using secondary electron detector...especially if the electrode is imaged before treatment with the plasma and then after treatment. Also, it may be possible to silver enhance ultra-small gold used for the ICC reaction. In this case you are looking at silver verses gold and should be able to distinguish the difference by backscattering or EDX mapping.

If I were You I would use FE-SEM work mode under low vacuum (dedicated to non-conductive samples).

Yes, Low vacuum could be helpful to minimize interference due to charge accumulation so that additional coating would not be necessary . However, the bulk of the sample is conductive. The plasma coat should be quite thin relative to the electrode and thus charge accumulation may not be a problem. High vacuum along with low voltage might do fine for secondary electron imaging. High vacuum images are almost always of higher resolution than those taken using low voltage. Also most high-resolution in-lens detectors require high vacuum for operation. Also charge effects can be minimized by using faster scans and averaging/integration when capturing the images.

The voltage level would be more problematic for back-scattering unless you are fortunate to have a low voltage backscatter detector. A light Pt (metal other than Au) coat might also work well if EDX mapping is used to differentiate plasma-covered and clear areas of the electrode. Normally I would also recommend C coating but that would interfere with identifying the plasma components in this case.

Very low voltage imaging using biased stages or other methods also help make imaging of very thin surface layers easier. However, these methods are not always available on user instruments. For those of you considering new microscopes, definitely look into these new modes of operation as they really do extend the versatility of your instruments.

Thank you Debra for the detailed information. Earlier I tried with low acceleration voltage (3 kV) but could not get any image except the dark area which is exposed to the plasma solution and bright area which is not.

Logudurai,

I do not know why you could not get an image as it may depend on your particular type of microscope or settings used. You would not be able to get a backscattered image with that low kV without a dedicated low voltage BS detector, but should be able to get a secondary electron image. You might want to contact one of the applications people from your instrument vendor to see if they could help you.

When I referred to low voltage above it was not just kV but also using stage biasing (or other methods used for enhanced charge reduction and imaging at extremely low voltages...often under 1kV) on uncoated nonconductive samples. These newer methods coupled with the appropriate detectors have added quite different capabilities than were previously available.

Cryo-SEM under low votage and AFM for morphology, Micro-raman and synchrotron STXM and infared for morpology and chemistry.