Forums
Bruker Media
Community Media
Bruker AFM Probes
SPM Digest
Application Notes
NanoTheater
Website
中文
Brochures & Datasheets
Publications
Probes Catalog
Events
Manuals & Documentation
Presentations
Guide to AFM Modes
News
Journal Club
Webinars & Video
Nanovations
Other
Hi,
I am used to imaged living cells with PeakForce Tapping over several hours. Keeping cells alive over that time is not a problem. Also keeping the same applied force is quite easy. The main problem is tip contamination. Even if the sample is perfectly clean, after scanning several hours, the tip automatically gets contaminated by (most likely) cells debris and the image gets more and more blury. I would like to know if anybody has an idea to improve that? The 2 things I could think of are:
1) Use hydrophobic probes. This would probably be the best but they don't give very good results with PFT (from what I observed), at least with soft cantilevers.
2) Use detergent at VERY low concentration. Of course not high-CMC detergent (that would also destabilize the cell membranes) but maybe fos-choline or this type of things.
Any input would be appreciated.
Thanks,
Alex.
Hi, Alex,
I could think of modifying your working tips to be hydrophobic (no need to worry about tip sharpness loss for cell imaging anyway), and a thin layer of PEG coating might worth a try, though I am not sure how it would affect the interaction between the tip and cell during scanning, your results will tell us I guess.
LA
Thanks for your prompt answer, Ang.
Actually the hydrophobic probes I tried were made of SAMs ended by -CH3. I didn't loose any resolution but got weird force curves. It was somtimes extremely hard to pull off.
PEG is super hydrophilic so it might interact a lot with the surface charges, especially at neutral pH, and stick to the cells. And I would have to make sure that it doesn't detach from the tip. This would also kill the cells. I will keep on searching.
Thanks!
Hydrophobic tip may not be a good choice then since it will introduce more complex tip-sample interaction and current algorism may not be able to deal with it.
We used to coat substrate surface with patterns of PEG coatings and it's very efficient to prevent cells attaching and force them to grow only in the PEG free regions. I believe PEG is a efficient blocker for nonspecific absorbsion of cell debris to tip surface.
OK, I trust you 100%. It seems like you have more experience than me on PEG. Did you publish on this? Or would you have any protocol to recommend?
Thanks in advance,
Two types of PEG you may try out. One is Methyl-PEG-X (one end with CH3 and the other end with any active functional group such as NHS or malemide or biotin etc depending on what chemical group on your tip). The other is PLL-g-PEG, we used this as glass surface coating and it works nicely (but it's noncovalent bonding so might degrade faster for tip coating). One small tip for PEG coating is the density, the higher density, the better anti-adhesion effect.
That's two good tricks we should have added to the Tech Note ;-)... Actually I would like to try both. Would you have any protocol or full publication? On a chemistry point of view, I understand how this works but having the protocol would help me save a lot of time.
I didn't include any because the publication or protocol I have seen or used were not for tip coating (we used for glass or PDMS and thus no need covalent bonding) or blocking purpose (we used two-end functional PEG as crosslinker to do single molecule force spectroscopy)... if you are still interested in, I can provide you some references.
I guess a brief protocol you can start with is to first have NH2 groups on your tip (easy to achieve folllowing your tip functionalization app note), then directly add in freshly dissolved Methyl-PEG-NHS following the product protocol (I usually chose from Pierce, you can find @ http://www.piercenet.com/browse.cfm?fldID=20CD10B3-1F10-4A87-B765-97DAB2FB6681), I guess longer PEG like PEG12 or PEG24 would achieve better blocking effect, but longer PEG may introduce extra interaction between tip and surface, so you may need careful tuning to see which one works better. Sorry no experience on this particular application so can't provide much more info.
I see what you mean. We call it heterobifunctional PEG linker.
Yes, having a tip coated with NH2 is pretty easy.
I never tried any chemical from Pierce but it seems to be a good brand.
I totally agree on your comments on the length of the linker. I think 12 carbons would be a good compromise. Anyway, I will never have time to test out all the combinations...
I have read your question. And I think maybe this page about cells (http://www.creative-biogene.com/Services/Stable-cell-line-generation) will help you in some way.