Sweers et al. Nanoscale Research Letters 2011, 6:270 http://www.nanoscalereslett.com/content/6/1/270 fibrils in contact mode using another probe (MSCT, tip E, 0.1 N/m, Veeco, Santa Barbara, CA, USA). This probe was selected to, on one hand, minimize the forces during contact mode imaging and, on the other hand, to match the spring constant of the cantilever to the stiff- ness of the sample for the indentation measurements. Every fibril was indented approximately 8 times at dif- ferent positions along its length. Prior to fibril indenta- tion, force curves were recorded on the mica substrate close to the fibril to determine deflection sensitivities of the cantilevers. Data analysis The raw deflection curves, obtained in the SPI mode, were converted to a force separation curve using the deflection sensitivities and the spring constants of the cantilevers in a custom written Matlab program. To extract the elastic modulus from the force separation curve, the Hertz model was used to analyze the force curve [39]. This model, in the case of a spherical inden- ter on a cylinder shaped object, is given in Figure 1 where F is the load, v the Poisson ratio, δ the separation, and E the modulus of elasticity. The equivalent contact radius Req for a spherical indenter with radius Rt, with an infinitely long cylinder with radius Rf is given by the expression in Figure 1. The modulus of elasticity was determined from the slope of the curve where F2/3 was plotted versus the separation. Small segments along this curve were fitted to a linear equation and the r2 value was determined for every fit, yielding an elastic modulus as a function of separation. From the point the force increases, the r2 value increases and only fits with an r2 above 0.95 were used in the analysis. From the point of contact the modulus of elasticity values for the following 2 nm were averaged (20% indentation [12]). Due to the finite thickness effects, the obtained modu- lus of elasticity is influenced by the stiff underlying sub- strate (mica). A correction factor for this effect was applied which was a function of the maximum applied force and the value of the uncorrected modulus of elas- ticity [16,36]. All analysis steps were implemented in a custom Matlab program. The algorithm analyzes both the force curve and the r2 curve to accurately determine the point-of-contact, that is, the separation at which the tip starts indenting the fibril. This point is defined as the point where the force distance curve leaves the baseline, and r2 adopts a value higher than 0.95. Harmonic force microscopy HarmoniX was performed under ambient conditions (that is, at room temperature without further control of humidity) on a Veeco Multimode microscope with a Nanoscope V controller (Veeco, Santa Barbara, CA, Page 9 of 10 USA). The analysis software uses the DMT model [40]. Torsional cantilevers (TL01, MikroMasch, Tallinn, Esto- nia) with a nominal spring constant of 2 N/m were used. The measured vertical and torsional resonance fre- quencies were 111 kHz and 1.1 MHz, respectively. The system was calibrated with a reference sample (model PS-LDPE, Veeco, Santa Barbara, CA, USA) [20]. Since HarmoniX assumes a spherical tip that indents an infi- nitely large and thick flat elastic surface, the value for the modulus of elasticity needs to be corrected offline. The first correction factor applied is to account for the differ- ent geometry, which in these experiments is a spherical tip indenting an infinitely long cylinder, see Figure 1. The correction factor used here is 2.1. The second correction factor was applied to account for the finite sample thick- ness of the protein fibril. A correction factor of 1.3, deter- mined by the SPI measurements, was used. Peakforce QNM Peakforce measurements were done on a Veeco Bio- scope Catalyst microscope with a Nanoscope V control- ler (Veeco, Santa Barbara, CA, USA). The analysis software uses the DMT model [40]. The measurements were done both in ambient conditions (uncontrolled humidity, temperature, and air pressure) and physiologi- cal buffer (10 mM Tris-HCl, 50 mM NaCl, pH 7.4). The manufacturer provides a list of optimal cantilevers to measure specific ranges of elastic moduli. For the ambi- ent measurements the stiff RTESP cantilevers (26.9 N/ m, Veeco, Santa Barbara, CA, USA) were used, due to the high adhesion forces observed for other, less stiff cantilevers. For the measurements performed in buffer we used a medium stiff cantilever: FMR-10 cantilevers (nominal spring constant 2.8 N/m, Nanoworld, Neuchâ- tel, Switzerland). Here, the elastic moduli are also cor- rected offline as described for the HarmoniX data (see Harmonic force microscopy). Image analysis Using SPIP software (Image Metrology A/S, Lyngby, Denmark), a trace was drawn on top of the fibril to determine the average height from the height images or modulus of elasticity from the stiffness maps of the indi- vidual fibrils, according to the procedure described in [4]. A point of potential confusion is that both Harmo- niX and Peakforce QNM create so-called ‘stiffness’ maps, which in the software is expressed in units of Pa. Technically this is not correct, since stiffness is expressed in units of N/m. The parameter in these images is a modulus of elasticity which is expressed in Pa. In this manuscript we therefore refer to these values as moduli of elasticity. All images in this article are line- wise corrected. Actual measurements are done on uncorrected images.