Sweers et al. Nanoscale Research Letters 2011, 6:270 http://www.nanoscalereslett.com/content/6/1/270 Page 4 of 10 Figure 3 Harmonic force microscopy images. Height (A, C) and corresponding elasticity images (B, D) of a-synuclein fibrils on mica. E represents the cross-sections drawn over the fibril in C. F represents the cross-section from D and shows a few scan artifacts. The background, mica, has here a stiffness of ± 1.5 GPa, probably caused by the limited range of elastic moduli which can be measured with the chosen cantilever. The peaks shown around 80 and 120 nm are edge effects caused by changing contact areas. The dip around 100 nm is assumed to be relevant for averaging and used to determine a modulus of elasticity. Scale bars are 250 nm. since one does not know the stiffness of the sample a priori, an estimation is necessary. This is also the case for the surface mapping methods. The nominal elastic modulus ranges accessible by HarmoniX and Peakforce QNM are 10 MPa-10 GPa and 0.7 MPa-70 GPa, respec- tively [18]. However, as noted above, this range depends on the cantilever that is used for the measurements and is in practice significantly smaller. A second point to consider when choosing the canti- lever is the adhesion between the tip and the sample. The spring constant of the cantilever should be suffi- ciently high to create enough force to come loose from the surface. In the PeakForce QNM experiments reported here on protein fibrils, performed in ambient air, an adhesion of few nanonewtons was observed. For reproducible and proper deflection curves in air we used in this case a cantilever with a spring constant of approximately 27 N/m. In the HarmoniX mode the fibrils are measured in a special tapping mode. In this mode reproducible results were obtained with cantile- vers with medium stiffness of 2 N/m in ambient condi- tions. The cantilever used for the nanoindentation measurements (0.1 N/m) showed an incredibly large artifact in both approach and retract curves at the 1 kHz ramp rate in Peakforce QNM in liquid, which was not seen in the nanoindentation measurements. This artifact could be induced by the impact of the effective mass and damping forces at the working