Hi friends,
Currently, I have to analyse leaf, stem and root tissues for physical presence of applied nano particles by foliar as well as soil. In that, I do not have much of idea about sample fixation for SEM/TEM analysis. SO kindly suggest me the protocol or procedure.
Thanks in advance.
In the past I have put them in a 2% glutaraldehyde solution in 0.2% PBS and kept them in the refrigerator overnight, then rinse with a 0.2% PBS solution, and then DI. That is probably sufficient for what you need to do, just dry them in an oven, possibly covered by a slide if you need to keep them flat (I used 60C for 12 hours). I was doing deposition on them, so I was very concerned about contamination, so I also did a 5% HCl soak, stirring with a stir bar until they change from green to brown, then rinse again in PBS and DI before drying. The HCl step may damage structures on the surface, so depending on what you are looking at, may not be the best option.
If you are interested only in finding nanoparticles on surface (with SEM), then the best way is to do nothing, just air dry you specimens. Treating with any liquids can wash out your particles. If you have good field emission SEM, do not coat your specimens, observe them at low voltage. If you interested in plant morphology also, to see if particles penetrate inside a plant (TEM), your task is more complicated. You need to fix you specimens, properly dehydrate them (in alcohol or acetone), embed in a resin and cut sections. Protocols are well developed and you can find plenty of them with Google.
Thank you Mr. Eric and Vladimir. If any protocol with reference is available,kindly provide.
Mr, Eric,
Actually i would like to see the physical presence of NPs in plant tissues like root and leaf tissue because I have applied NPs by the mode of foliar as well as soil. So that, I am in need of clear protocol to perform the analysis by SEM/TEM.
For SEM collect samples with foliar spray and without foliar spray. Clean the surface with double distilled water 4-5 times and keep it in cool dry condition. Cut them into the circle shape with lesser the size of Stud and assess through SEM-EDAX Micro analysis report and compare the both.
Dr. Manikandan,
- In this case, fixation will be done by some preservative like formaldehyde and osmiun oxide etc. I do not know clearly.
- In one reference, they were mentioned that cut the leaf by fine blade. And not mentioned clearly. thats why am asking?
Dear Gopi, don't use any preservatives, it will contaminate your sample, it shows higher concentration of elements H,C,and O in your micro analysis report and also final interpretation could be difficult. Clean the sample with only double distilled water and for sectioning leaves use stain steel knife.The same we had done for our sample.
if you are using sem, fixation is a must or the sample would give you a 'not so pretty' picture (biological sample can easily shrink ). i fix it with glutaraldehyde 4% > washing with phosphate buffer solution > post fix with osmium tetraoxide(not necessarily for sem but for tem is a must) > washing again with PBS > dehydration with a series of alcohol 30%, 50%, 75%, 95%, 100% > critical point drying >mounting > coating > view. hope this help.
hello, i am facing the same problem, so i will be grateful for the solution also.
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