Sweers et al. Nanoscale Research Letters 2011, 6:270 http://www.nanoscalereslett.com/content/6/1/270 of 39% calculated above makes interpreting these results very difficult. Conclusions The nanometer scale diameters of a-synuclein protein fibrils pose some serious challenges for interpretation of the data obtained with SPI, HarmoniX and Peakforce QNM. The typical size scales of the fibrils give rise to finite sample thickness effects [16,36]. Furthermore, these fibrils cannot be described as a flat film on a sur- face for which all the standard models are valid [39,40]. Finally, these samples have strong adhesive properties which results in choosing cantilevers that possibly result in less contrast between the fibrils and the surface, because of the mismatch between cantilever and sample stiffness. All these difficulties are addressable with the conventional nanoindentation measurements, where the analysis is mostly done offline and in custom-written algorithms. For the surface property mapping techniques it is at this point only possible to customize the analysis in a limited manner. The methods come with specific conditions in which the analysis is valid. First, the tip should be a hard sphere compared to the sample. Sec- ond, only elastic deformation is taken into account. Last, the sample should not be confined vertically (finite sample thickness effect) or laterally (by surrounding material) [18]. For protein fibrils the second condition is not actually known, after indentation with high forces (> 3 nN) the fibrils appear to be broken, while with lower forces they stay intact (< 2 nN). The third condition is not met in case of the protein fibrils. For HarmoniX it is also good to keep in mind that theoretically one needs an infinite number of frequency components to reconstruct the real time interaction between the tip and the surface [18]. To obtain in a short amount of time quantitative modulus of elasticity for protein fibrils the surface prop- erty methods are relatively easy to use and fast. How- ever, recording individual curves on the fibrils during scanning is necessary to analyze the curves for all the conditions that are not met in these methods. In case of the measurements done on the protein fibrils the differ- ences are within each others error ranges. This may not be the case for other biological structures. It is essential to understand the limitations of each method and care- fully analyze the data, including the individual force curves, according to the valid conditions for the specific structures. Methods Sample preparation E46K disease mutant a-synuclein was recombinantly expressed and purified as previously described [4]. A 100 μM monomeric E46K solution in 10 mM Tris-HCl, Page 8 of 10 50 mM NaCl, pH 7.4 was incubated at 70°C in Eppen- dorf tubes under constant shaking. After 27 h, well- defined protein fibrils were formed in solution, which was verified by a Thioflavin T fluorescence assay specific for cross-beta structures characteristic of amyloid fibrils. Samples for AFM imaging in liquid were prepared by placing 50 μl of a 5× diluted solution containing fibrils on the mica substrate. This solution was allowed to adsorb for 10 min and then washed gently with 200 μl buffer. For imaging, 80 μl of fresh buffer solution was placed on the sample. We used the same buffer solution (10 mM Tris-HCl, 50 mM NaCl, pH 7.4) for both dilu- tion and imaging. For the measurements performed in ambient air, a 10× diluted protein solution was placed on mica substrates and allowed to adsorb in the same manner as described above. Subsequently, the sample was washed with 200 μl milliQ water and dried with a gentle nitrogen stream. AFM cantilever and tip characterization The tip radius was determined with two different meth- ods. First, from the AFM height images of protein fibrils the tip radius was derived from the fibril height-to- width ratio based on tip-sample convolution [21,31]. Only fibrils that were perpendicular to the scan axis were used. From the tip sample convolution method an average tip radius of 100 nm was determined. Second, the tip was imaged by scanning electron microscopy (Philips XL30 ESEM-FEG). With the SEM, the average tip radius was found to be approximately 80 nm. For both methods, the tip resulted in a considerably larger number than the nominal tip radius provided by the manufacturer. An average value of 90 nm was used in the analysis with an error of 30%. The cantilever spring constants were determined with the thermal noise method implemented in the Veeco software and were assumed to have a 5% error [33]. Single-point nanoindentation A Bioscope II microscope (Veeco, Santa Barbara, CA, USA) was used for the SPI experiments. In order to measure the fibril heights, AFM tapping mode images were recorded in a physiological buffer (10 mM Tris- HCl, 50 mM NaCl, pH 7.4) in tapping mode with low force settings (reduced to 3 nm, 80-90% of the free amplitude) to minimize interaction with the sample. We use silicon nitride probes (MSCT, tip F, 0.5 N/m, Veeco, Santa Barbara, CA, USA) for these measurements. The average fibril height measured in tapping mode is used to determine the surface contact area for all three indentation methods. The indentation measurements were performed with the “Point and Shoot” application in the NanoScope 7.30 (Build R2Sr1.) software. To locate the indentation locations we first imaged the