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I am a relatively new AFM user and I am looking for advice on using probes with extremely low spring constants (in the range or 0.01 - 0.05 N/m). My long term goal is to image red blood cells (RBCs) using the AFM and observe structural changes as they encounter different proteins. I have tried imaging RBCs using the basic ScanAsyst Fluid probes with limited success. I think the spring constant is too large on these tips so I'm trying to use something much smaller, like the MSNL probes. Before I attempt a living cell, however, I would like to be able to image a simple piece of glass that has a hole in it roughly 3um wide. I have had plenty of success imaging these pieces of glass in both air and fluid using the ScanAsyst probes. I have never used the MSNL probes before but I'm having a hard time getting good force curves.
My question is two fold:
1. Does anyone have any advice for imaging stiff surfaces with low spring constant probes? Is that even practical and/or possible?
2. Does anyone have any advice on imaging RBCs and what type of probes they think would work the best?
Thank you in advance for your help!
Joshua
Thank you for your response. We have had success imageing in PeakForce Tapping mode using the MLCT Probes - A Cantilever. We found that too soft of a spring constant tended to produce a poor image but that if the probes were too stiff the cells would come off of the surface they were stuck to. Thanks again for your help
Hi Joshua,
Imaging living cells is never easy but RBCs are one of the stiffest existing eukaryotic cells. The main problem is to find a way to attach them on the surface without changing their physical or chemical properties. If you overcome that step (and it seems like you did), and you operate in the proper near-physiological buffer imaging RBCs should not be that challenging, especially by using Peak Force Tapping. One of the difficulty is to correctly adjust the scanning parameters to get an optimal tracking on BOTH glass and cells. If you operate in PFT, especially PFQNM, the critical step is to get good force curves to get a good deflection sensitivity. For this, it's strongly advised to work on the peak force setpoint and the gain. For RBCs, the setpoint can be increased to (once calibrated) 1nN but the gain HAS to be as low as possible. Selecting AUTO GAIN ON with ScanAsyst is a good option. After thermal Tune, you should find a spring constant close to the nominal value given by the manufacturer. If you have a deviation < or > to 50%, the defl. sens. might be wrong, then you shold ramp again until you get decent force curves. Another way to check that is too make a High Speed Data Capture (HSDC) and analyze it with Nanoscope Analysis software: on the deflection channel, if the glass part is much more noisy than the cell part, this might also be due to a wrong calibration which can lead to inverted contrasts, especially in the DMT modulus channel.
Now coming back to the choice of probes, I think SCANASYST FLUID (not SCANASYST FLUID PLUS!) probes can definitely used to image living RBCs. They have a k = 0.7 N/m which is still in the right range. some MLCT probes can also be used for PFT operation: A (0.07), C (0.01) and D (0.03 N/m) are too soft but E (0.1) and F (0.6) should work.
If you want to try in regular tapping mode, MLCT E and F are recommended. If you prefer contact mode or Force Volume, I recommend MLCT C and D.
I don't know what your degree of exertise in AFM is. If my comments are not clear enough, you can contact me directly:
alexandre.berquand@bruker-nano.com
Good luck!
Alex.
I preplied to this post a while ago but now that the technique has greatly improved, I can strongly recommend PeakForce Tapping to image such samples:
ScanAsyst-Fluid probes can be used to image a very wide range of biological samples, down to bacteria and even "not too soft" eukaryiotic cells. If your cells are softer than 50kPa, I also recommend OBL probes. The OBL-B is better than the A because it's longer so you are less likely to get scanning artifacts but because of the low spring constant (0.006 N/m, which actually makes it the softest existing commercial probes) you might have to wait a while (typically 30 min) before engaging.
Dear J Price,
Good to hear that you managed to get good images. Also keep posted because in early/mid August will be released an Application Note fully dedicated to PFT and PFQNM on soft biological samples. We also have a webinar on that topic on August 17.
Hope this will help out.
Best,