Sweers et al. Nanoscale Research Letters 2011, 6:270 http://www.nanoscalereslett.com/content/6/1/270 Page 3 of 10 Figure 2 Typical force curves. (A) A typical force versus piezo displacement curve obtained from the measurement, with the approach curve (solid red) and the retract curve (dashed blue). (B) Force versus separation approach curve calculated from the force versus piezo displacement curve. (C) Force to the power of 2/3 versus separation approach curve, showing distinct transition from the tip only sensing the fibril (part I) to the part where the tip is sensing the mica under the fibril (part II) until the part where the tip is only pressing on the mica (part III). From the slope of part I, a modulus of elasticity of 1.2 GPa was calculated for the force curve presented here. Harmonic force microscopy A sample of a-synuclein fibrils deposited on mica was scanned. Figure 3 shows two typical images recorded, with corresponding height and elasticity profiles. The fibrils show considerably lower modulus of elasticity compared to the background. However, the edges of the fibril show increased modulus of elasticity values, also displayed in the cross-section of the fibril shown in Figure 3F. We attribute this effect is due to the chan- ging contact area compared to the contact area shown in Figure 1 where the tip is indenting the middle of the fibril. This artifact is also visible in the height images derived from the harmonic force mode, shown in Figure 3E, and they are therefore not used in further analysis. For each individual fibril, the values for the elastic mod- ulus measured along the fibril were averaged. The aver- age value was 1.2 ± 0.2 GPa (N = 95). Peakforce QNM The surface property mapping technique Peakforce QNM is able to image the sample both in ambient con- ditions and in buffer solution. Figure 4 shows height images and the corresponding elasticity maps obtained with Peakforce QNM of a-synuclein fibrils, obtained in buffer (Figure 4A, B) and in air (Figure 4C, D). These images were obtained with a high setpoint of around 15 nN and show that for both liquid and ambient condi- tions the height and elasticity ranges which can be obtained with Peakforce QNM are similar. However, this large setpoint causes the fibrils to break, especially in liquid, see Figure 4A. To prevent damage to the a-synuclein fibrils, a lower setpoint of 1-2 nN was used. This resulted in intact fibrils with significant lower values of the elastic moduli (Figure 5). The elastic modulus for each fibril is deter- mined from the average value of the DMT modulus obtained along the fibril length. This resulted in a mod- ulus of elasticity of 1.3 ± 0.3 GPa (N = 57) for the fibrils in ambient conditions and 1.0 ± 0.2 GPa (N = 59) for those in liquid. Discussion Choosing the right cantilever In order to measure the elastic properties of a material, the choice of the cantilever is key. In nanoindentation the highest sensitivity (and thus accuracy) is achieved if the spring constant of the probe cantilever is identical to the effective spring constant of the sample (also referred to as contact stiffness), see Figure 6. If the spring constant of the cantilever is more than 10 times lower or higher than that of the sample, the sensitivity is about 3 times lower, see Figure 6 making the determi- nation of the elastic modulus less accurate. Practically