I have chitosan-TPP nanoparticles, prepared by probe sonication. Basically, I add 4 mL of TPP solution (1.76 mg/ml) to 10 mL chitosan solution (3mg/ml). Then I probe sonicate it (5 min; 30-10 on/off). Finally I separate the NPs by centrifugation (15000 rpm, 20 min, room temp).
I get quite good DLS results (app 250 nm size; 0.1 PDI). The problem is, when I try to get SEM image, I get micrographs as attached (either with the ddH20 resuspended pellet or with NP suspension obtained right after the probe sonication).
I air dry the NP (1:100 dilution) directly on SEM stub. What is that background? It is also observed on the NP as well? Did anyone observe such a micrograph? What should I do? Really appreciate your help. Thank you in advance
Have you coated the sample with something conductive before the SEM examination?
The particles and background have the same fine texture. That is very common effect when the sample is overcoated. For example, just gold grains when the sputter was not good or coating time was too long. Another option - something in the suspension (monomers, etc), that contaminates the surface.
P.S. Many years ago I studied some artificial virus-like particles. We didn't have a good SEM around, but conventional TEM with negative staining (like for viruses) was working very well.
please check the SEM images of following paper :
Article Porous chitosan by crosslinking with tricarboxylic acid and ...
Check around, search, google and follow a well-known, well-referenced, much used procedure for preparing the sample for SEM examination. It happens with excess coating or layering, which has been observed by many researchers.
Thank you very much for the answers. Denis Korneev and Riaz A. Khan we coated the samples with gold (8-10 nm) is it too much? would 4 nm be enough? Denis Korneev I also though about the unreacted monomer as well, but this micrograph is after centrifugation step (where I thought that I can get rid of the unreacted chitosan/TPP). In the case that I cannot remove the excess monomers, would dialysis work? I will definitely go for TEM as well.
Sina Safavi the SEM images in the paper is just like what I got. But these are hydrogels, may be my nanoparticle preparation procedure is not efficient to form good NPs.
Could you try to view the sample uncoated? The image quality will be lower but it might show you if you are looking at gold (alternatively see if someone could coat with platinum or another metal that gives a finer grain size). I see you have an FEI FEGSEM. if it is an ESEM you could try the 5kV cone and large field detector. You could also try the GSED detectors. If it is not an ESEM you could try 2kV or lower with the ETD. It will be difficult and you may not be able to to resolve the features. Good luck.
Deniz Sezlev , no worries. 8-10 nm of gold is too much for such magnification. Should be not more than 5 nm (it is better to use Pt or Ir than gold, because they provide finer grains).
An image of an overcoated sample is attached. Looks pretty similar.
Good luck!
Denis Korneev, thank you so much. Very helpful image. I will make it less coated. I hope they have Pt. Thank you so much, again.
@David Patton, the FESEM is not in our facility, I am sending it someplace else to take the image and unfortunately they don't know to use the ESEM module (although they have it). I hope they can coat it with Pt and I will tell them to try with 2kv. Thank you very much
Dear Deniz Sezlev
I think hydrogel may have formed in your experiment because of both chitosan and TPP are hydrophile then I suggest you to try remove all water molecules by drying under vacuum and also remove extra TPP. On the other hand I guess maybe your particles are over coated so this SEM will observed hence you would try particle synthesis with lower amount of TPP. I hope that I have could help you.
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