Breakthrough instruments and products: DriveAFM for high-performance atomic force microscopy.

Atomic force microscopy is a powerful technique for measurement and mapping of nanoscale topography and electrical and mechanical sample properties. The Nanosurf DriveAFM is a new generation instrument that combines ease of use and high performance through full motorization, CleanDrive photothermal excitation, and a mechanical and electrical design that allows for both high-resolution and large-range imaging.

Reference-free alignment and sorting of single-molecule force spectroscopy data.

Single-molecule force spectroscopy has become a versatile tool for investigating the (un)folding of proteins and other polymeric molecules. Like other single-molecule techniques, single-molecule force spectroscopy requires recording and analysis of large data sets to extract statistically meaningful conclusions. Here, we present a data analysis tool that provides efficient filtering of heterogeneous data sets, brings spectra into register based on a reference-free alignment algorithm, and determines automatically the location of unfolding barriers. Furthermore, it groups spectra according to the number of unfolding events, subclassifies the spectra using cross correlation-based sorting, and extracts unfolding pathways by principal component analysis and clustering methods to extracted peak positions. Our approach has been tested on a data set obtained through mechanical unfolding of bacteriorhodopsin (bR), which contained a significant number of spectra that did not show the well-known bR fingerprint. In addition, we have tested the performance of the data analysis tool on unfolding data of the soluble multidomain (Ig27)(8) protein.

Atomic force microscopy of biological membranes.

Atomic force microscopy (AFM) is an ideal method to study the surface topography of biological membranes. It allows membranes that are adsorbed to flat solid supports to be raster-scanned in physiological solutions with an atomically sharp tip. Therefore, AFM is capable of observing biological molecular machines at work. In addition, the tip can be tethered to the end of a single membrane protein, and forces acting on the tip upon its retraction indicate barriers that occur during the process of protein unfolding. Here we discuss the fundamental limitations of AFM determined by the properties of cantilevers, present aspects of sample preparation, and review results achieved on reconstituted and native biological membranes.

Conductive supports for combined AFM-SECM on biological membranes.

Four different conductive supports are analysed regarding their suitability for combined atomic force and scanning electrochemical microscopy (AFM-SECM) on biological membranes. Highly oriented pyrolytic graphite (HOPG), MoS(2), template stripped gold, and template stripped platinum are compared as supports for high resolution imaging of reconstituted membrane proteins or native membranes, and as electrodes for transferring electrons from or to a redox molecule. We demonstrate that high resolution topographs of the bacterial outer membrane protein F can be recorded by contact mode AFM on all four supports. Electrochemical feedback experiments with conductive cantilevers that feature nanometre-scale electrodes showed fast re-oxidation of the redox couple Ru(NH(3))(6)(3+/2+) with the two metal supports after prolonged immersion in electrolyte. In contrast, the re-oxidation rates decayed quickly to unpractical levels with HOPG or MoS(2) under physiological conditions. On HOPG we observed heterogeneity in the re-oxidation rate of the redox molecules with higher feedback currents at step edges. The latter results demonstrate the capability of conductive cantilevers with small electrodes to measure minor variations in an SECM signal and to relate them to nanometre-scale features in a simultaneously recorded AFM topography. Rapid decay of re-oxidation rate and surface heterogeneity make HOPG or MoS(2) less attractive for combined AFM-SECM experiments on biological membranes than template stripped gold or platinum supports.

High-throughput single-molecule force spectroscopy for membrane proteins.

Atomic force microscopy-based single-molecule force spectroscopy (SMFS) is a powerful tool for studying the mechanical properties, intermolecular and intramolecular interactions, unfolding pathways, and energy landscapes of membrane proteins. One limiting factor for the large-scale applicability of SMFS on membrane proteins is its low efficiency in data acquisition. We have developed a semi-automated high-throughput SMFS (HT-SMFS) procedure for efficient data acquisition. In addition, we present a coarse filter to efficiently extract protein unfolding events from large data sets. The HT-SMFS procedure and the coarse filter were validated using the proton pump bacteriorhodopsin (BR) from Halobacterium salinarum and the L-arginine/agmatine antiporter AdiC from the bacterium Escherichia coli. To screen for molecular interactions between AdiC and its substrates, we recorded data sets in the absence and in the presence of L-arginine, D-arginine, and agmatine. Altogether ∼400 000 force-distance curves were recorded. Application of coarse filtering to this wealth of data yielded six data sets with ∼200 (AdiC) and ∼400 (BR) force-distance spectra in each. Importantly, the raw data for most of these data sets were acquired in one to two days, opening new perspectives for HT-SMFS applications.

Characterization of microfabricated probes for combined atomic force and high-resolution scanning electrochemical microscopy.

A combined atomic force and scanning electrochemical microscope probe is presented. The probe is electrically insulated except at the very apex of the tip, which has a radius of curvature in the range of 10-15 nm. Steady-state cyclic voltammetry measurements for the reduction of Ru(NH3)6Cl3 and feedback experiments showed a distinct and reproducible response of the electrode. These experimental results agreed with finite element simulations for the corresponding diffusion process. Sequentially topographical and electrochemical studies of Pt lines deposited onto Si3N4 and spaced 100 nm apart (edge to edge) showed a lateral electrochemical resolution of 10 nm.