If so any hints would be appreciated. I find adhesive forces too great in air and am unable to make sufficient contact in liquid. The only method I have found to work at all is by fixing the gels and the dehydrating in a graded alcohol series, however this is quite laborious and also I don't know what it does to fibre orientation.
Dear Thomas,
Imaging collagen hydrogels has been one of my connundrum over the last few years and we now have decided to image them air dried / compressed etc.. as it is quite painful to image them in liquid due to the pore size in between the fibrils. We have had mild success with various AFM based approach to image such hydrogel and therefore i would advise now to use 2-photon or SICM to image the hydrogel. Of course it all depends on what you want to see. However, we perform all the AFM mechanical analysis in liquid and have a robust protocol to do so. The alcohol dehydration will not affect your fibrils' orientation however it may impact a little bit on the fibril diameter as it removes interstitial water.
Any questions dont hesitate to ask.
But unfortunately "soft" cantilever is not guarantee success and sample can be destroyed. For example, we analyzed surface of peptone agar and after this see deepening it done by the cantilever (figure attached).
I know that, the well tuned parameters are critical. I succeeded in this type of study continuously since 5 years before. It is difficult for beginners.
I am using alginate hydrogels. Do you suggest I should dry them? I already ordered low spring constant cantilevers. Any other suggestion? Many thanks
Dear Laurent Bozec,
Have you worked also with alginate hydrogels?
Do you suggest to perform force-distance curves in liquid? (then I should change the setip) Because I only have nice AFM (in contact in air) signal after my gels are dehydrated (with air) and I am not sure if that is correct.
Should I also try with etOH?
I would really appreciate any advice because I don't have experience.
Best, and thanks, Ana
Dear Maria,
We have not worked with alginate gel but i had a small experience in imaging such alginate with optical coherence tomography. I was bemused me at the time observing how upon hydration the gel not only contracted up a lot but also the pore size in the gel varies enormously. Small pores became very sizable. As it was a one-off experiment testing the imaging methods, this behaviour was not investigated further. The contraction was not something that i expected!
However, if you are going to rehydrate alginate gel, please be mindful of the internal structure of the gel, especially if you want to undertake AFM mechanical measurement. The principle of FD on bhydrogel does not however change regardless of the type of gel:
However, if you are going to rehydrate alginate gel, please be mindful of the internal structure of the gel, especially if you want to undertake AFM mechanical measurement. The principle of FD on hydrogel does not however change regardless of the type of gel:
- mount the gel so that when hydrated, it does not move (or slide) with respect to the glass-slide or petri-dish. Using glue can have adverse effect. I would recommend a mechanical clamping approach. The gel will expand/contract and you need to account for that in your clamping.
- adjust the size of the bead use for functionalization of the cantilever to exceed the pore size of your gel. If this is not possible (i.e. the pores are too big) - you will spend a good amount of time "landing" the probe in area suitable for measurement. This can be frustrating!
- start with the smallest indentation load as possible and ramp up the contact force until you observe an adequate indentation. You do not want to bury you probe into the gel on firs contact! Beware of the thermal drift as you will likely need to adjust the contact force to compensate for the probe/sample change in contact force as a result of the shrinking/expansion of the sample. It is not uncommon to leave the hydrated sample mounted on the AFM for 10 to 30min to rest before performing the force-distance curve.
All in all FD in liquid is not trivial but can be achieved with great control and care. FD in air is very easy in comparison. Understanding how your sample behave when rehydrated is a huge part of the success.
Hope this helps - if not send me an email
Best,
Laurent@
Dear Laurent,
Thank you very much for all these details!
You are very kind. I will work on the details you mentioned,
Thank you again,
Ana
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