Sweers et al. Nanoscale Research Letters 2011, 6:270 http://www.nanoscalereslett.com/content/6/1/270 At this point the tip is retracted again. During this approach and retract cycle the force is continuously measured, resulting in a force versus distance graph. AFM nanoindentation has been performed on different biological substrates such as collagen [15], insulin fibrils, and crystals [16], but also on different polymeric materi- als, such as fibrils used for biodegradable scaffolds [17]. The approach-retract cycle is typically performed at a rate of 0.5 to 10 Hz, which makes this method inher- ently slow. To get an overview of the mechanical prop- erties of a sample, nanoindentation can be used in a force-volume mode. Here, for every pixel in an image a complete force curve is recorded, which results in data acquisition times of up to hours for a single image. Recently, several different surface property mapping techniques have become available that work at much higher speeds, leading to significantly increased data throughput [18-20]. Two commercially available approaches are PeakForce QNM and Harmonic force microscopy or HarmoniX (Veeco, Santa Barbara, CA, USA). PeakForce QNM is based on the force-volume approach; however, the speed of taking the force curves is significantly increased (either at 1 or 2 kHz). In this mode the maximum force exerted on the sample is maintained constant, which is beneficial for soft delicate biological samples. Because of the recent introduction of the Peakforce QNM method, only a few studies have been reported, such as the stiffness mapping of polymer blends [18]. HarmoniX is another surface property mapping techni- que based on the nonlinear dynamic behavior of a canti- lever in tapping mode due to repulsive and attractive forces caused by the specific material characteristics of the sample acting on the tip [21,22]. Because of the low bandwidth of the cantilever response, this information ends up in the phase image as obtained during tapping mode imaging. This phase signal is related to energy dis- sipation, which is determined by the viscoelastic and adhesive properties of the sample [21,23]. However, because of the convolution of multiple physical proper- ties into one signal, interpretation of these images is not straightforward. The higher harmonic vibrations of the cantilever excited by these material properties can pro- vide more information, but they are heavily suppressed and are difficult to measure [21,24]. In HarmoniX, a tor- sional cantilever with the tip positioned off-axis solves this problem and acts as a high bandwidth force sensor [24]. HarmoniX has been applied to both polymers and biological features, for example, DNA [25,26]. We used these three different methods, SPI, PeakForce QNM, and HarmoniX, to determine the modulus of elasticity of protein nanofibrils, generated from the E46K mutant of the human a-synuclein protein. The resulting values for the elastic modulus are in the range Page 2 of 10 between 1.3 and 2.1 GPa. We discuss the relative merits of the application of these three methods specifically for the determination of the elastic properties of protein fibrils in more detail, with particular emphasis on the differences and difficulties of each method. Results Single-point nanoindentation experiments in liquid a-Synuclein fibrils deposited on mica were scanned both in tapping mode and contact mode, respectively, for determining the height and finding the indentation points for the SPI measurements. We determined an average fibril height of 9.0 ± 0.4 nm (N = 60) from the tapping mode images. This average height value was used to determine the effective contact surface in the indentation measurements according to the model shown in Figure 1. The fibril heights measured in con- tact mode imaging were considerably lower and were therefore not used in determining the average fibril height. This was attributed to the pressure from the tip on the sample. The force exerted on fibrils with the 0.1 N/m cantilever during scanning was between 0.5 and 1 nN. We performed nanoindentation experiments on five fibrils, each of which was indented 8 times at different locations along its length. A typical force distance curve resulting from this procedure is shown in Figure 2A. The absence of adhesion during the measurements allowed the use of the Hertz model. Although every fibril was indented 8 times, not all curves were suitable for analysis. For some curves, the r2 values of the linear fit did not exceed 0.95 in the part where the tip was indenting the fibril (part 1 in Figure 2C). From the curves that were analyzed, an average elastic modulus of 1.3 ± 0.4 GPa (N = 31) was found for a-synuclein fibrils. Figure 1 Schematic representation of equivalent contact radius. Schematic representation of the AFM tip as a spherical indenter and the protein fibril as an infinitely long cylinder.