P. L. T. M. Frederix

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.

Quantitative dynamic-mode scanning force microscopy in liquid,

We describe a method to perform dynamic-mode scanning force microscopy in liquid with true atomic resolution. A frequency-modulation technique is used to maintain constant amplitude, phase, and frequency shift of the cantilever oscillation. As a consequence, the tip-sample interaction force is well defined and quantitative. The force sensitivity is demonstrated by imaging and deliberate bending of a peptide loop connecting transmembrane helices of the membrane protein bacteriorhodopsin. The experimental setup allows further enhancement of the force sensitivity by the use of small cantilevers.

A Fabry–Perot interferometer for micrometer-sized cantilevers

We have developed a Fabry–Perot interferometer detecting the deflection of micrometer-sized cantilevers and other micromechanical devices, at a working distance of 0.8mm. At 1MHz, a noise floor of 1fm∕Hz is obtained. The detector is mounted on a piezo motor for three-axis alignment. The angular alignment is not critical. The interferometer can be operated in vacuum, air, and liquid. It is particularly suited for scanning force microscopy with small cantilevers, or with larger cantilevers simultaneously monitoring vertical and lateral forces.

Assessment of insulated conductive cantilevers for biology and electrochemistry

This paper describes the characterization and application of electrically insulated conductive tips mounted on a cantilever for use in an atomic force microscope and operated in liquid. These multifunctional probes were microfabricated and designed for measurements on biological samples in buffer solution, but they can also be employed for electrochemical applications, in particular scanning electrochemical microscopy. The silicon nitride based cantilevers had a spring constant ≤0.1 N m-1 and a conductive tip, which was insulated except at the apex. The conductive core of the tip consisted of a metal, e.g. platinum silicide, and exhibited a typical radius of 15 nm. The mechanical and electrical characterization of the probe is presented and discussed. First measurements on the hexagonally packed intermediate layer of Deinococcus radiodurans demonstrated the possibility to adjust the image contrast by applying a voltage between a support and the conductive tip and to measure variations of less than 1 pA in faradaic current with a lateral resolution of 7.8 nm.

Symmetrically arranged quartz tuning fork with soft cantilever for intermittent contact mode atomic force microscopy

A self-sensing and -actuating probe for dynamic mode atomic force microscopy (AFM) based on a commercial quartz tuning fork and a microfabricated cantilever is presented. The U-shaped cantilever, exhibiting a sharp tip, is combined with the tuning fork in a symmetrical arrangement, such that each of the two legs of the cantilever is fixed to one of the prongs of the tuning fork. The tuning fork is used as an oscillatory force sensor. Its frequency and amplitude govern that of the tip vibration, while the cantilever determines the spring constant of the whole probe. The frequency of the tip vibration for AFM operations can be much higher than the resonance frequency of the cantilever. A probe comprising a silicon nitride cantilever (0.1 N/m) is used to image monoatomic terraces of graphite in the intermittent contact mode. A much softer cantilever (0.01 N/m) is used to analyze the topography of a microelectronic chip in the same mode. Moreover, a bacterial surface layer hexagonally packed intermediate layer of Deinococcus radiodurans is imaged in a buffer solution. The tip vibration was again generated by the tuning fork while the sample interaction was measured using the standard optical detection scheme in this experiment. These probes are suited for batch fabrication and assembly and, therefore, enlarge the applications for the tuning fork based AFM.

Development of Insulated Conductive Probes with Platinum Silicide Tips for Atomic Force Microscopy in Cell Biology

A microfabrication process of a multifunctional probe is introduced for atomic force microscopy and various electrochemical measurements on biological samples in buffer solution. The silicon nitride probes have a spring constant lower than 0.1 N/m and a conductive tip, which is tightly insulated except at the apex. The conductive core of the tip consists of PtxSi y and shows a typical radius of curvature of 15 nm. A simultaneous measurement of topography and electrical current on graphite in air was demonstrated.

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.

Force-conductance correlation in individual molecular junctions

Conducting atomic force microscopy is an attractive approach enabling the correlation of mechanical and electrical properties in individual molecular junctions. Here we report on measurements of gold-gold and gold-octanedithiol-gold junctions. We introduce two-dimensional histograms in the form of scatter plots to better analyze the correlation between force and conductance. In this representation, the junction-forming octanedithiol compounds lead to a very clear step in the force-conductance data, which is not observed for control monothiol compounds. The conductance found for octanedithiols is in agreement with the idea that junction conductance is dominated by a single molecule.

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.

Insulated Conductive Probes for in situ Experiments in Structural Biology

We report about the development of a multifunctional AFM probe, which allows to perform simultaneous measurements of topography and electrical properties of biological samples in a buffer solution. The quality of the AFM probes has been experimentally assessed by acquiring topography measurements of bacteriorhodopsin membranes in buffer solution and topographical and electrical current images of HOPG in air.