Sweers et al. Nanoscale Research Letters 2011, 6:270 http://www.nanoscalereslett.com/content/6/1/270 However, even when using blunt tips there are mea- surement errors which have to be considered. The radius of the tip in indentation studies is often deter- mined by scanning electron microscopy [16] after the indentation experiments where the tip shape could be influenced by wear [30]. Another method is to deter- mine the tip radius from tip sample convolution models [21,31]. From previous studies the error from the tip sample convolution method is around 30% [32]. An additional important step is the calibration of the cantilever spring constant. There are a number of tech- niques available to determine the spring constant, each with their own uncertainties [33-35]. In this study, can- tilevers were calibrated with the thermal noise method, which has an associated average error of 5% [33]. All three methods described here are susceptible to relatively large systematic errors arising from the com- pounding of errors inherent to the different calibration and characterization techniques. Using the law of propa- gation of errors, we estimate that this systematic error combined with the above-mentioned and the previously described 2% error in the deflection sensitivity measure- ments [34] yields an uncertainty of approximately 39% of the average measured value. In addition to these errors, experimental data are also influenced by the expected statistical variation due to heterogeneity of a large sample set. Finite sample thickness When indenting small fibrillar features with a relatively large tip radius one has to take the finite sample thick- ness effects into account. As shown in Figure 2C the force curve displays distinct regimes: from being free in air above the sample to the initial fibril indenting sec- tion where the tip ‘only’ feels the fibril (Figure 2C, part I), to where the mica underneath starts to play a role (Figure 2C, part II), and finally to the last section where only the hard surface is felt by the tip (Figure 2C, part III). The initial 20% of the total height of a feature is thought to be unaffected by these final sample thickness effects for large objects relative to the tip radius [12]. However, at the typical size scales of these nanofibrils, a correction for these effects is necessary [16,36]. In the Peakforce QNM software these effects are not consid- ered and therefore not compensated for [18]. In Harmo- niX there is also no correction for these effects. However, the question is whether these effects are nearly as pronounced in HarmoniX because of the small indentations that are made with this technique. In the SPI measurements from this study the correction factor was small (~1.3) because of the rather high modulus of elasticity. Discussion of results All methods used in this study yielded moduli of elasti- city between 1.3 and 2.1 GPa (see Table 1), which is Table 1 Overview of results from different methods Method Environment Operation frequency (Hz) Nanoindentation Liquid Peakforce QNM Liquid Peakforce QNM Air HarmoniX Air 1 103 103 105 Page 7 of 10 Uncorrected modulus of elasticity (GPa) - 1.0 ± 0.2 1.3 ± 0.3 1.2 ± 0.2 Modulus of elasticity (GPa) 1.3 ± 0.4 1.6 ± 0.3 2.1 ± 0.5 1.9 ± 0.3 Overview of results from different methods under different environmental conditions, 4th column represents the corrected values for modulus of elasticity. This correction is the same as done in the analysis of the indentation measurements; the contact area is changed to a spherical indenter on an infinite cylinder (average fibril height of 9.0 ± 0.4 nm, measured in AFM tapping mode) and is corrected for the finite sample thickness as described in the “Methods” section. close to values found for collagen and other amyloid fibrils [10,15]. These values are somewhat smaller than those obtained for films made with fibrillar networks of b-lactoglobulin (5.2-6.2 GPa) and lysozyme (6.7-7.2 GPa) [37], and potentially reflect the differences in experimental conditions. We also measured insulin and lysozyme amyloid fibrils using HarmoniX under ambient conditions. The values measured, 1.4 ± 0.2 GPa for lyso- zyme fibrils and 1.4 ± 0.1 GPa for insulin, were com- mensurate to that measured for a-synuclein. The modulus previously found for insulin fibrils measured with SPI in liquid, which is at a lower working speed, is three orders of magnitude lower [16]. However, Smith et al. [11] have found a value of 3.3 GPa for insulin fibril using force spectroscopy on suspended fibrils. Note that in this work all the methods result in relatively similar values, although they all have very different operation speeds. The spread in the SPI measurements is also compar- able to earlier work. The reason for this spread, besides the previously mentioned errors, has been related to heterogeneity in the internal packing of amyloids [1,16,38]. Both surface contact area and finite sample thickness corrections were performed offline on the HarmoniX and Peakforce QNM data, see Table 1 which results in higher values. The finite sample correction value found in the analysis of the SPI of 1.3 and the relation for a spherical indenter on an infinite long cylinder are used. The high modulus of elasticity of the fibrils suggests a high packing density. The difference between liquid and ambient air conditions becomes more significant after correction. With the uncorrected values the difference is lower, which suggests little room for water within the fibril, but the corrected results could point to an observed drying effect. However, the large spread, seen in Table 1 and in pre- vious studies, combined with the large systematic error