Bridging the Titin Gap The muscle protein titin is a molecular spring that has been extensively studied by single-molecule unfolding experiments and by molecular simulation. However, experimental and simulated unfolding could not be compared directly because they differ by orders of magnitude in pulling velocity. Rico et al. (p 741 ) developed high-speed force spectroscopy to pull titin molecules at speeds that reach the lower limits of molecular dynamics simulations. Bridging the gap between simulation and experiment clarified the mechanism of conformational changes in titin.

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Nanoscale electron transport through the purple membrane monolayer, a two-dimensional crystal lattice of the transmembrane protein bacteriorhodopsin, is studied by conductive atomic force microscopy. We demonstrate that the purple membrane exhibits nonresonant tunneling transport, with two characteristic tunneling regimes depending on the applied voltage ͑direct and Fowler-Nordheim͒. Our results show that the purple membrane can carry significant current density at the nanometer scale, several orders of magnitude larger than previously estimated by macroscale measurements. DOI: 10.1103/PhysRevE.76.041919 PACS number͑s͒: 87.80.Ϫy, 73.40.Rw, 85.65.ϩh The purple membrane ͑PM͒ is a two-dimensional crystal lattice naturally present in the cell membrane of Halobacterium salinarum. It is composed of a lipid bilayer and a single-protein species, the Bacteriorhodopsin ͑bR͒, in a lipidto-protein ratio of 10 ͑mol/ mol͒. Bacteriorhodopsin acts as a light-driven proton pump, converting solar energy into an electrochemical proton gradient across the cell membrane ͓1,2͔. Its functional stability under different environmental conditions combined with easy and large production has made bR a model protein for studies of charge transport on cell membranes, as well as an excellent candidate for bioelectronic applications ͓3,4͔.Despite its enormous interest, only a few studies regarding the electron transport measurements of a single PM layer have been reported so far, leading to an incomplete and controversial picture ͓5-7͔. The main obstacle encountered in measuring the electrical conductivity of the PM monolayer ͑ϳ5 nm thick͒ resides on providing reliable electrical contact at the electrode-membrane interface. Measured currents can dramatically differ by orders of magnitude from measurement to measurement on supposedly identical conditions, being extremely sensitive to the electrode-membrane distance as well as the applied load on the membrane. To date, two methods have been reported: ͑i͒ measuring the current of a monolayer confined between two submillimeter-sized electrodes ͓5͔ and ͑ii͒ probing the nanoscale conductivity of the monolayer using a scanning tunneling microscope ͑STM͒ ͓6,7͔. The millimeter-sized electrode configuration demands a flat and hole-free monolayer covering the entire electrode surface, which is, however, difficult to fabricate. Furthermore, averaging of biological information on a macroscale level is inherent to this method. To study electron conduction at the molecular level, scanning probe techniques are by far the most appropriate approach. STM, however, has an intrinsic limit in the tunneling current feedback for insulating samples which impedes the control and quantification of the probe-membrane distance and forces applied on the biomolecules.In this article we use conductive atomic force microscopy ͑C-AFM͒ as an extremely controlled method to provide a comprehensive and unambiguous model of electron conduction in the PM monolayer. Conductive AFM has demonstrated to be well suited to studying th...

Nanoscale capacitance imaging with attofarad resolution (∼1 aF) of a nano-structured oxide thin film, using ac current sensing atomic force microscopy, is reported. Capacitance images are shown to follow the topographic profile of the oxide closely, with nanometre vertical resolution. A comparison between experimental data and theoretical models shows that the capacitance variations observed in the measurements can be mainly associated with the capacitance probed by the tip apex and not with positional changes of stray capacitance contributions. Capacitance versus distance measurements further support this conclusion. The application of this technique to the characterization of samples with non-voltage-dependent capacitance, such as very thin dielectric films, self-assembled monolayers and biological membranes, can provide new insight into the dielectric properties at the nanoscale.

A simple method to measure the static dielectric constant of thin films with nanometric spatial resolution is presented. The dielectric constant is extracted from DC electrostatic force measurements with the use of an accurate analytical model. The method is validated here on thin silicon dioxide films (8 nm thick, dielectric constant approximately 4) and purple membrane monolayers (6 nm thick, dielectric constant approximately 2), providing results in excellent agreement with those recently obtained by nanoscale capacitance microscopy using a current-sensing approach. The main advantage of the force detection approach resides in its simplicity and direct application on any commercial atomic force microscope with no need of additional sophisticated electronics, thus being easily available to researchers in materials science, biophysics and semiconductor technology.

Membrane proteins diffuse within the membrane, form oligomers and supramolecular assemblies. Using high-speed atomic force microscopy, we present direct experimental measure of an in-membrane-plane interaction potential between membrane proteins. In purple membranes, ATP-synthase c-rings formed dimers that temporarily dissociated. C-ring dimers revealed subdiffusive motion, while dissociated monomers diffused freely. C-rings center-to-center distance probability distribution allowed the calculation and modeling of an in-membrane-plane energy landscape that presented repulsion at 80 Å, most stable dimer association at 103 Å (-3.5 k(B)T strength), and dissociation at 125 Å (-1 k(B)T strength). This first experimental data of nonlabeled membrane protein diffusion and the corresponding in-membrane-plane interaction energy landscape characterized membrane protein interaction with an attractive range of several k(B)T that reaches to a radius of ∼50 Å within the membrane plane.

Biological atomic force microscopy (AFM) is a fast growing and advancing field. This review's objective is to overview the state of the art and to retrace achievements of biological AFM as presented by past and present research, and wishes to give a (subjective) outlook where AFM may go in the upcoming years. The following areas of interest are discussed: High-resolution imaging, cell imaging, single molecule force spectroscopy, cell mechanical measurements, combined AFM instrumentation, and AFM instrumentation. Of all these topics, particular representative examples are shown, each of them standing for a variety of achievements by many research groups.

High-speed atomic force microscopy is a powerful tool for studying structure and dynamics of proteins. So far, however, high-speed atomic force microscopy was restricted to well-controlled molecular systems of purified proteins. Here we integrate an optical microscopy path into high-speed atomic force microscopy, allowing bright field and fluorescence microscopy, without loss of high-speed atomic force microscopy performance. This hybrid high-speed atomic force microscopy/optical microscopy setup allows positioning of the high-speed atomic force microscopy tip with high spatial precision on an optically identified zone of interest on cells. We present movies at 960 ms per frame displaying aquaporin-0 array and single molecule dynamics in the plasma membrane of intact eye lens cells. This hybrid setup allows high-speed atomic force microscopy imaging on cells about 1,000 times faster than conventional atomic force microscopy/optical microscopy setups, and allows first time visualization of unlabelled membrane proteins on a eukaryotic cell under physiological conditions. This development advances high-speed atomic force microscopy from molecular to cell biology to analyse cellular processes at the membrane such as signalling, infection, transport and diffusion.