I am trying to measure stiffness of red blood cells in RPMI using AFM force spectroscopy method. In literature, the typical indentation of RBC at a force of 500pN is 500nm. But in my case I see values of 500pN for 50nm. I am using MLCT cantilever from Bruker which has a spring constant of 30pN/nm on Picoscan 3000 Molecular Imaging AFM. Due to this, the fit to Hertz model gives me values around 300kPa instead of reported value of 1kPa. What could be the reason for such a low indentation and high forces?
Literature Ref: J. L. Maciaszek and G. Lykotrafitis, “Sickle cell trait human erythrocytes are significantly stiffer than normal,” J. Biomech. (in press). 10.1016/j.jbiomech.2010.11.008
Dear Sumanth, how did you calculate the elastic constant of the probe and the deflection sensitivity?
You can check this methodological work for example. DOI: 10.1038/s41598-017-05383-0
Best
Massimiliano
The following material seems to be useful to find your real answer.
L. Waite and J. Fine, Applied Biofluid Mechanics (McGraw-Hill Co., USA, 2007).
Have you tried different cantilevers? The interaction force between the tip and the sample strongly depends on the tip curvature radius R. Maybe the literature data and your data were obtained with cantilevers which have different R?
By the way, you shouldn't always believe the manufacturers data on the cantilever radius. Any typical cantilever can become blunt or dirty during the experiment - it will lead to a change in the cantilever radius.
Have you tried colloid probes? They can be useful. Our paper https://www.jbiomech.com/article/S0021-9290(13)00064-X/fulltext describes some facts about it.
When you choose a cantilever, you should use a rectangular one rather than a triangular one. You can easily calibrate a rectangular cantilever using Sader method.
Good luck!
Hi Massimilano Galluzzi,
I have gone through the paper you suggested for methodology. It is very exhaustive and gives clear idea about the procedure to get consistent results.
The use of vibrometer to measure the accurate force constant seems to be the key point which gives a correction factor. This is mandatory for conversion of deflection to nm. But I don't have vibrometer and the cantilevers we are using are not pre-calibrated by the manufacturer. I see the effect which I explained in my question with both spherical (Hydra6V-100NG, AppNano with 5-9um PS bead, thermal calibrated force constant 0.4N/m) and pyramidal tips (MLCT-D, Bruker with 20-50nm tip radius, thermal calibrated force constant 0.03N/m). Which of these cantilevers is supposed to be better? And is there any way to get better results without using vibrometer?
Dear Sumanth Kumar, sorry for late answer. In my experience I prefer spherical probes for mechanical measurements, especially if you are working in liquid on bio samples. Another solution, in place of vibrometer, could be a set of calibrated samples (especially with modulus in the range of your research samples). Recursively using SNAP you can obtain k and zsens quite precisely. For example I used a set of flat and soft (1 - 30 kPa) hydrogels calibrated by macroscopic rheology. We figured out that spherical probes have a superior performance for nanomechanics, while sharp probes are slightly overestimating modulus. You can also buy calibrated samples (you can find PDMS from Bruker for example).
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