Nanoscale Structural and Mechanical Properties of Alpha-Synuclein Amyloid Fibrils

In this thesis we have studied the structural and mechanical properties of amyloid fibrils formed by the human (alpha)-synuclein protein, associated with Parkinson's disease

Spatially resolved requency-dependent elasticity measured with pulsed force microscope and nanoindentation

Recently several atomic force microscopy (AFM)-based surface property mapping techniques like pulsed force microscopy (PFM), harmonic force microscopy or Peakforce QNM® have been introduced to measure the nano- and micro-mechanical properties of materials. These modes all work at different operating frequencies. However, complex materials are known to display viscoelastic behavior, a combination of solid and fluid-like responses, depending on the frequency at which the sample is probed. In this report, we show that the frequency-dependent mechanical behavior of complex materials, such as polymer blends that are frequently used as calibration samples, is clearly measurable with AFM. Although this frequency-dependent mechanical behavior is an established observation, we demonstrate that the new high frequency mapping techniques enable AFM-based rheology with nanoscale spatial resolution over a much broader frequency range compared to previous AFM-based studies. We further highlight that it is essential to account for the frequency-dependent variation in mechanical properties when using these thin polymer samples as calibration materials for elasticity measurements by high-frequency surface property mapping techniques. These results have significant implications for the accurate interpretation of the nanomechanical properties of polymers or complex biological samples. The calibration sample is composed of a blend of soft and hard polymers, consisting of low-density polyethylene (LDPE) islands in a polystyrene (PS) surrounding, with a stiffness of 0.2 GPa and 2 GPa respectively. The spring constant of the AFM cantilever was selected to match the stiffness of LDPE. From 260 Hz to 1100 Hz the sample was imaged with the PFM method. At low frequencies (0.5–35 Hz), single-point nanoindentation was performed. In addition to the material's stiffness, the relative heights of the LDPE islands (with respect to the PS) were determined as a function of the frequency. At the lower operation frequencies for PFM, the islands exhibited lower heights than when measured with tapping mode at 120 kHz. Both spring constants and heights at the different frequencies clearly show a frequency-dependent behavior

Characterizing nanoscale morphologic and mechanical properties of α-synuclein amyloid fibrils with atomic force microscopy

We have used atomic force microscopy (AFM) to image wild-type and disease-related mutant α-synuclein (aS) amyloid fibrils deposited on various hard and soft surfaces, ranging from freshly cleaved mica to two different supported lipid bilayers: phosphatidylcholine (POPC) and a mixture of POPC and phosphatidylglycerol (POPG). Quantitative morphologic analyses show that fibrils deposited on freshly cleaved mica, gold and glass substrates appeared to have similar heights and lengths, suggesting that the surface–fibril interaction in these cases does not influence the fibril morphology. When the same fibril sample is deposited on HOPG or quartz, the aS fibrils appear shorter in length and completely distorted, respectively, indicating that the interaction with these surfaces severely affects the fibril morphology. Sequentially recorded AFM images of fibrils on POPC bilayers clearly revealed that the amyloid fibrils were mobile on the bilayer, indicating that the fibrils are in thermal equilibrium on the surface. Persistence lengths of the fibrils on mica and POPC have been determined using two different methods, and reveal no significant differences, indicating that, although the fibrils appear immobile on mica, they do thermally equilibrate in 2D before they finally attach to the substrate. The persistence length of aS fibrils is found to be between 3.3 and 7.1 μm, which is within the expected range for amyloid fibrils. We observe no fibril movement on the mixed POPC/POPG bilayer, suggesting a stronger interaction with the lipids. Furthermore, the reduced heights of the fibrils on top of the mixed bilayer suggest that the fibrils are partly embedded within the bilayer