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Hi,
I am a new user to the AFM technology. Currently, I am trying to use a tipless cantilever to measure the stiffness of a hollow thin shell microsphere (following the protocol of a published article: JE McKendry, CA Grant, BRG Johnson, et al.; Bubble Science, Engineering and Technology; 2010 (2) 48-54). The microsphere is immobilized on a microscope slide coated with poly-L-lysine (PLL). When I preform the deflection sensitivity calibration, I use a clean slide (coated with PLL) with no microsphere samples. Is this correct? I read in other forum answers that I am supposed to use a "hard" surface. Does that mean I should use a slide WITHOUT PLL coating?
At this moment, I am getting a stiffness value that is higher than expected. Since it is assumed that the cantilever and the microsphere act like two springs in series. The measured effective stiffness should be in this relationship to the cantilever spring constant: 1/keff = 1/kc + 1/ksphere. Hence I can calculate the stiffness of the microsphere. However, the effective stiffness values I got so far are all higher than the cantilever spring constant kc (obtained using built-in thermal tuning), which made the final value of ksphere have no physical meaning. I am not sure if this discrepancy is caused by my cantilever deflection sensitivity calibration done on a PLL coated surface, rather than a true "hard" surface.
Thank you very much for your help.
Hi -
using the tipless cantilever method can be really tricky. In particular, the effective spring constant of the cantilever is proportional to the cube of the length, so the spring constant if you push with the very end will be significantly different than if the contact point is a few microns in from the end. Additionally, you have to account for the angle of alignment between the optics and the cantilever in backing out the spring constant. There's a paper about it, although I can't seem to find the reference in my records - sorry.
As for the PLL coating, I don't know if that could be significant. A glass slide should be hard enough to get a good sensitivity calibration, but without knowing more about the PLL coating and how thick it is, I can't comment on whether that would effect the calibration.
That's a very hard question because the answer strongly depends on the thickness of the coating and also the tip radius. Do you have any rough idea about the thickness? About 10 years ago, I made an interesting experiment: I coated a composite polymer sample with different thicknesses of gold (I know a metal layer is very different from a polymer layer but the way ot affects the value of the measured stiffness is similar): 1, 2, 5, 10, 20 and 100 nm and each time captured phase images in tapping mode to see how the phase contrast between the 2 components varies. At a moderate ratio setpoint, even a thickness of 2 nm is enough to induce a spectacular change in mechanical properties! I can forward you the paper if you are interested.
I guess that with a polymer, the effect will be less dramatic but strictly speaking the deflection sensitivity should be updated on a clean (which means pure) and very stiff material. Glass is OK but it's even better to do it on an even less compliant sample like mica or silicon, or quartz.
A last point: One might argue that whatever the thickness of the polymer, the mechanical properties will be the same. The tip will just go trough and hit the glass. This is not true. I don't have any precise reference in mind but it has been demonstrated that depending on their concentration on a surface, certain polymers can exhibit very different physical behavior.
Anyway, in your case I think the layer is rather thin so I don't expect a huge change in mechanical properties. I would say that only a VERY picky reviewer would mind... If I ever happen to review your work, I will never hassle you on such a point! ;-)...
Alex.