Hello everyone,
I am trying to measure Young's modulus on plant cell walls. I am usin atomic force microscopy to do it. I tried fixing my cells with poly-l-lysine and it does attach some cells, except that cells keep moving on one side or slide whenever the cantilever is scanning them. Anyone has an idea about how to fix the whole cell file ad keeping it from moving while scanning?
Thank you
Dear Fatima,
I don't think, its the poly-Lysine coating which helps fixing cells. For mammalian cells, poly-Lysine is used to help the cells to attach. Once, they are attached they can crawl around or stay in place dependent on the cell type. You can try to fix cells gently with 2% cold paraformaldehyde (after brief boiling to allow for polymerization), which preserves structures pretty nicely.
Best, Daniel.
Dear Fatima: wet or dry mode of AFM recording? Thank you for clarification.
Hello Wolfgang, I am trying to do wet mode AFM..but I'll try any suggestions..Thank you
Dear Fatima: what kind is the surface [e.g. glass, or other...] you try to fix your plant cell wall material now....do you use "sections" of plant cells or e. g. use "homogenate" ....Please inform about the technique you use for now a bit more in detail....just to be able to imagine and to know where you are coming from.... (BTW: have you done a Literature search already?....Apologize for my asking the latter....)
Dear Wolfgang :
I have done literature search already and asked some of the authors for some advice. Most people do AFM on whole tissu or organs, and in dry medium. I am trying to do it on Arabidopsis thalilana cell suspension in liquid conditions. The difficulty is that I need to keep the cell surface attached on one side of the glass coverslip while the other side is free to be examined with the AFM cantilever in a liquid medium. I am currently trying to apply a small amount of liquid low-melt agarose, put my cells, let dry and the gel will probably be solidified with the cells on some places, and then I will apply liquid medium...If it works, it will be great, I'm still looking for suggestions...I will try with these few modifications this afternoon, next time will be next week...
I also tried a simple and naive method by applying my cells to a double-sided scotch tape, it works for the mechanical side, but the cell wall is rapidly modified because of the cell mortality.
Once I master the 'attachemnet' of the cells, I will try different conditions, for ex :
w/o inducing plamolysis...
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