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Trying to get force curves on collagen, but having trouble

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lmoore38 posted on Mon, Jul 11 2011 10:53 AM

Hello, I am a graduate student at Saint Louis University working with the AFM.  I am trying to get force curves on a collagen hydrogel over a period of 5 days, while a capillary network develops.  Right now, I am just trying to get a force curve on my collagen sample with no cells.  We have a Novascan machine, and I am using spherical tips with a spring constant of 0.6 N/m. I am running into a few problems that I was hoping I might find an answer to here.

1.  We were finding that when trying to position the probe over the sample, the very back of the cantilever was submerging into the collagen, thus not allowing the actual tip to function properly.  To solve this, I used less collagen on the coverslip, and tried to position the probe so that just the very tip was over the collagen itself.  We did get a force curve that looked similar to those in literature, but I'm not sure if it is actually reading the glass underneath the collagen.  We are so close to the edge, that I'm wondering if I'm reading collagen or glass beneath it since the sample is so thin at the edge. 

2.  When we tried to position the probe just a little further in to read a thicker part of the gel, we are finding that the jump-to-contact forces are so strong, that even if the back of the probe doesn't submerge, the tip itself is sucked into the gel and can't come out.  We've tried withdrawing a few microns each time, but the same thing keeps happening. 

Is my sample too soft?  Do you think these tips are ok?  Do I need to fix the sample if I want any good readings?  I was thinking that submerging the sample in PBS or something would help eliminate those jump-to-contact forces that seem to be messing up my data, but wanted to get a few professional opinions on if this is a good idea or not. 

Thank you so much for your time!

Lisa

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Answered (Not Verified) Ang Li replied on Mon, Jul 11 2011 10:41 PM
Suggested by Ang Li

Hi, Lisa, here are some tips that may help you:

1. to get rid of glass substrate effect, you'd better have at least ~10 um thick collagen layer and only fit ~100nm-500nm indentation depth in your force curve. if your sample is soft and thick, try not to engage before running force curve, instead, manually adjust the position of your cantilever to a few um above the surface and run the force curve with a ramp size larger than that distance. though I am not sure whether your system allows you doing so, Bruker's point & shoot function can achieve that and you can find an application note somewhere in this website.

2. you already have the answer, run this exp in liquid will reduce most of the jump-to-contact force as well as strong adhesion due to meniscus force. this will help significantly if these two are not your interests.

you have to evaluate whether fixation will change the properties of collagen that you are interested and wanna measure~ 

Good luck and feel free to post any other questions if you have!

LA 

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