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.     

AFM-based force spectroscopy measurements of mature amyloid fibrils of the peptide glucagon

We report on the mechanical characterization of individual mature amyloid fibrils by atomic force microscopy (AFM) and AFM-based single-molecule force spectroscopy (SMFS). These self-assembling materials, formed from the 29-residue amphiphatic peptide hormone glucagon, were found to display a reversible elastic behaviour. Based on AFM morphology and SMFS studies, we suggest that the observed elasticity is due to a force-induced conformational transition which is reversible due to the β-helical conformation of protofibrils, allowing a high degree of extension. The elastic properties of such mature fibrils contribute to their high stability, suggesting that the internal hydrophobic interactions of amyloid fibrils are likely to be of fundamental importance in the assembly of amyloid fibrils and therefore for the understanding of the progression of their associated pathogenic disorders. In addition, such biological amyloid fibril structures with highly stable mechanical properties can potentially be used to produce nanofibres (nanowires) that may be suitable for nanotechnological applications.     

Molecular orientation and its influence on autocorrelation amplitudes in single-molecule imaging experiments

The concentration dependence of autocorrelation amplitudes obtained from orientationally fixed single molecules is explored using theory, simulation, and experimental results. Autocorrelation amplitudes obtained under such circumstances are shown to be approximately 2-fold larger than predicted in previous studies (Koppel, D. E. Phys. Rev. A 1974, 10, 1938-1945 and citing references), which frequently assume polarization-independent excitation and detection. A detailed derivation of the autocorrelation amplitude expected under conditions frequently employed in single-molecule experiments is given. Simulated and experimental single-molecule image data obtained from samples incorporating fixed single molecules are used to verify the correctness of the model. These results are compared to both simulated and experimental time transient data in which the molecules exhibit predominantly fast rotational reorientation and to which previously reported models apply. The experimental results employed are obtained from dye-doped mesoporous silica thin films studied at different levels of hydration. The theory and results obtained are of importance to the determination of molecular concentrations from single-molecule image and time transient autocorrelation data, in situations where the molecules exhibit permanent or reversible adsorption at fixed orientations in or on thin-film materials.     

Nanoengineering a single-molecule mechanical switch using DNA self-assembly

The ability to manipulate and observe single biological molecules has led to both fundamental scientific discoveries and new methods in nanoscale engineering. A common challenge in many single-molecule experiments is reliably linking molecules to surfaces, and identifying their interactions. We have met this challenge by nanoengineering a novel DNA-based linker that behaves as a force-activated switch, providing a molecular signature that can eliminate errant data arising from non-specific and multiple interactions. By integrating a receptor and ligand into a single piece of DNA using DNA self-assembly, a single tether can be positively identified by force-extension behavior, and receptor-ligand unbinding easily identified by a sudden increase in tether length. Additionally, under proper conditions the exact same pair of molecules can be repeatedly bound and unbound. Our approach is simple, versatile and modular, and can be easily implemented using standard commercial reagents and laboratory equipment. In addition to improving the reliability and accuracy of force measurements, this single-molecule mechanical switch paves the way for high-throughput serial measurements, single-molecule on-rate studies, and investigations of population heterogeneity.     

Dissecting contact mechanics from quantum interference in single-molecule junctions of stilbene derivatives

Electronic factors in molecules such as quantum interference and cross-conjugation can lead to dramatic modulation and suppression of conductance in single-molecule junctions. Probing such effects at the single-molecule level requires simultaneous measurements of independent junction properties, as conductance alone cannot provide conclusive evidence of junction formation for molecules with low conductivity. Here, we compare the mechanics of the conducting para-terminated 4,4'-di(methylthio)stilbene and moderately conducting 1,2-bis(4-(methylthio)phenyl)ethane to that of insulating meta-terminated 3,3'-di(methylthio)stilbene single-molecule junctions. We simultaneously measure force and conductance across single-molecule junctions and use force signatures to obtain independent evidence of junction formation and rupture in the meta-linked cross-conjugated molecule even when no clear low-bias conductance is measured. By separately quantifying conductance and mechanics, we identify the formation of atypical 3,3'-di(methylthio)stilbene molecular junctions that are mechanically stable but electronically decoupled. While theoretical studies have envisaged many plausible systems where quantum interference might be observed, our experiments provide the first direct quantitative study of the interplay between contact mechanics and the distinctively quantum mechanical nature of electronic transport in single-molecule junctions.     

Error analysis of the rapid lifetime determination method for double-exponential decays and new windowing schemes

The rapid lifetime determination method (RLD) is a mathematical technique for extremely rapid evaluations of lifetimes in exponential decays. It has been applied in luminescence microscopy and single-molecule lifetime evaluation. To date, the primary application has been in single-exponential evaluations. We present extensions of the method to double exponentials. Using Monte Carlo simulations, we assess the performance of both the double-exponential decay with known lifetimes and the double-exponential decay with unknown preexponential factors and lifetimes. Precision is evaluated as a function of the noise level (Poisson statistics), the ratios of the lifetimes, the ratios of their preexponential factors, and the fitting window. Optimum measurement conditions are determined. RLD is shown to work well over a wide range of practical experimental conditions. If the lifetimes are known, the preexponential factors can be determined with good precision even at low total counts (10(4)). With unknown preexponential factors and lifetimes, precisions decrease but are still acceptable. A new gating scheme (overlapped gating) is shown to offer improved precision for the case of a single-exponential decay. Theoretical predictions are tested against actual experimental data from a laser-based lifetime instrument.     

Molecular resolution of cell adhesion forces

Recently, AFM-based force spectroscopy has been used to quantify single-molecule adhesion forces on living ameboid cells. Force spectroscopy was used to measure the rupture forces of single receptor-ligand bonds which can occur rapidly between the cell types used, a metastasising B16 melanoma cell and a vascular bEnd.3 endothelial cell. Parameters which influence the critical experimental conditions are discussed to discriminate between multiple bond ruptures and single bonds. Under physiological conditions of temperature and pH the force measurements show an average rupture force of 33 pN (SD = 12 pN) for single bonds. Single-molecule force spectroscopy will be very useful to study the regulation of cell adhesion on a molecular level in normal processes, such as leukocyte homing, and in major human disorders, including tumor metastasis, autoimmune diseases and atherosclerosis.     

Statistics of single-molecule surface enhanced Raman scattering signals: fluctuation analysis with multiple analyte techniques

The mathematical background, based on a variation of the principal component analysis (PCA) method, is developed for the understanding of fluctuating multiple analyte single-molecule (SM) surface enhanced Raman scattering (SERS) signals; with emphasis on the bianalyte SERS technique developed recently. The method and its significance are presented to provide a systematic framework with which several aspects of the statistics of SM-SERS signals can be analyzed in general. We also apply the method to a concrete example of bianalyte statistics in silver colloidal solutions and discuss related topics around experimental issues and the interpretation of single-molecule SERS data.     

预应力混凝土桥梁徐变分析的全量形式自动递进法

首先将预应力混凝土桥梁中考虑收缩、徐变影响的任意时刻混凝土应力、应变关系在持荷时段内写成代数形式,引入内力平衡方程及变形协调条件后,提出了计入截面上钢筋位置、配筋率、预应力钢筋松弛、混凝土弹性模量随时间变化等影响的徐变效应分析的全量形式自动递进法,并建立了计算式,适用于任何形式的收缩、徐变特性表达式;基于建立的全量形式公式,可方便地求解任意时刻混凝土、钢筋的应力与应变和梁体竖向变形。理论分析与试验结果比较表明,公式可方便地控制计算精度,直至给出满意的结果。将计算式编程后极易与目前桥梁设计中常用的杆系有限元软件接口,进行可靠的徐变分析。     

Liquid-Phase Electron Microscopy for Soft Matter Science and Biology

Innovations in liquid-phase electron microscopy (LP-EM) have made it possible to perform experiments at the optimized conditions needed to examine soft matter. The main obstacle is conducting experiments in such a way that electron beam radiation can be used to obtain answers for scientific questions without changing the structure and (bio)chemical processes in the sample due to the influence of the radiation. By overcoming these experimental difficulties at least partially, LP-EM has evolved into a new microscopy method with nanometer spatial resolution and sub-second temporal resolution for analysis of soft matter in materials science and biology. Both experimental design and applications of LP-EM for soft matter materials science and biological research are reviewed, and a perspective of possible future directions is given.     

Application of the TraPPE Force Field to Predicting Isothermal Pressure–Volume Curves at High Pressures and High Temperatures

Knowledge of the thermophysical properties of materials at extreme pressure and temperature conditions is essential for improving our understanding of many planetary and detonation processes. Significant gaps in what is known about the behavior of materials at high density and high temperature exist, largely, due to the limitations and dangers of performing experiments at the necessary extreme conditions. Modeling these systems through the use of equations of state and particle-based simulation methods significantly extends the range of pressures and temperatures that can be safely studied. The reliability of such calculations depend on the accuracy of the models used. Here we present an assessment of the united-atom version of the TraPPE (transferable potentials for phase equilibria) force field and single-site exp-6 representations for methane, methanol, oxygen, and ammonia at extreme conditions. As shown by Monte Carlo simulations in the isobaric–isothermal ensemble, the TraPPE models, despite being parameterized to the vapor–liquid coexistence curve (i.e., relatively mild conditions), perform remarkably well in the high-pressure/high-temperature regime. The single-site exp-6 models can fit experimental data in the high-pressure/temperature regime very well, but the parameters are less transferable to conditions below the critical temperature.     

Simultaneous,hybrid single-molecule method by optical tweezers and fluorescence

As studies on life sciences progress toward the single-molecule level, new experiments have put forward more requirements for simultaneously displaying the mechanical properties and conformational changes of biomolecules. Optical tweezers and fluorescence microscopy have been combined to solve this problem. The combination of instruments forms a new generation of hybrid single-molecule technology that breaks through the limitations of traditional biochemical analysis. Powerful manipulation and fluorescence visualization have been widely used,and these techniques provide new possibilities for studying complex biochemical reactions at the singlemolecule level. This paper explains the features of this combined technique, including the application characteristics of single-trap and dual-traps, the anti-bleaching method, and optical tweezers combined with epifluorescence, confocal fluorescence, total internal reflection fluorescence, and other fluorescence methods.Using typical experiments, we analyze technical solutions and explain the factors and principles that instrument designers should consider. This review aims to give an introduction to this novel fusion technology process and describe important biological results.     

Apertureless near-field/far-field CW two-photon microscope for biological and material imaging and spectroscopic applications

We present the development of a versatile spectroscopic imaging tool to allow for imaging with single-molecule sensitivity and high spatial resolution. The microscope allows for near-field and subdiffraction-limited far-field imaging by integrating a shear-force microscope on top of a custom inverted microscope design. The instrument has the ability to image in ambient conditions with optical resolutions on the order of tens of nanometers in the near field. A single low-cost computer controls the microscope with a field programmable gate array data acquisition card. High spatial resolution imaging is achieved with an inexpensive CW multiphoton excitation source, using an apertureless probe and simplified optical pathways. The high-resolution, combined with high collection efficiency and single-molecule sensitive optical capabilities of the microscope, are demonstrated with a low-cost CW laser source as well as a mode-locked laser source.     

Nanoindentation of viscoelastic solids: A critical assessment of experimental methods

The viscoelastic functions measured by nanoindentation (or atomic force microscopy), are coupled in complex ways to the measurement system’s time constants, phase shifts created by the instrument’s electronics, the actuator’s dynamics, the instrument’s load frame stiffness and requisite modeling assumptions. The ways in which these factors potentially affect the load, displacement, phase angle, stiffness and damping data are discussed in the context of nanoindentation experiments performed in the time and frequency domains. By drawing attention to these potential sources of experimental error, the objective is to motivate experimental verification in a manner that will enhance accuracy and, thus, enable future breakthroughs in the application of nanoindentation to viscoelastic solids.     

Real-Time Digital Multifunction Instrument for Power Quality Integrated Indexes Measurement

In this paper, the design and implementation of a high-performance, real-time power quality (PQ)-measuring instrument based on a digital signal processor is discussed. The system is realized according to the latest standards about PQ monitoring, and it can be reconfigured for future developments of the standards. The instrument is particularly implemented to operate in stand-alone mode, and it is interfaced with a hardware module that adopts Transmission Control Protocol/Internet Protocol to exchange information and data. The main advantage of the realized device is the possibility to perform a comprehensive PQ monitoring that adopts a single low-cost instrument. Finally, this paper reports some preliminary experimental results of the accuracy characterization of an implemented prototype of the proposed instrument.      

Protein Experiment: Scientific Data Processing Platform for On-Flight Experiment Tuning

In 2009, the PROTEIN experiment was run on board of the Columbus module of the International Space Station to investigate the crystallization of proteins in microgravity using the Protein Crystallization Diagnostics Facility. This instrument was designed to allow almost real time modification of remote-operated space experiments on request by the Science Team. The complexity of the experiments and the expected high volume of raw scientific data required the development of ad-hoc analysis tools in order to provide scientist with a quick and in-depth picture of experiment progress. The integrated data analysis platform presented in this paper allowed early inspection of running experiment results and provided information for impromptu, fine-tuning experimental conditions. The feedback loop starting with remote data acquisition, followed by on ground analysis performed by the platform and finishing with experiment redesign and update, is described through several examples.     

Constitutive modeling of aluminum nitride for large strain,high-strain rate,and high-pressure applications

This paper presents constitutive modeling of aluminum nitride (AlN) for severe loading conditions that produce large strains, high-strain rates, and high pressures. The Johnson–Holmquist constitutive model (JH-2) for brittle materials is used. Constants are obtained for the model using existing test data that include both laboratory and ballistic experiments. Due to the wide range of experimental data the majority of constants are determined explicitly. The process of determining constants is provided in detail. The model and constants are used to perform computations of many of the experiments including those not used to generate the constants. The computational results are used to validate the model, provide insight into the response of AlN, and to demonstrate that one set of constants can provide reasonable results over a broad range of experimental data.     

Correlation-matrix analysis of two-color coincidence events in single-molecule fluorescence experiments

We introduce a robust and relatively easy-to-use method to evaluate the quality of two-color (or more) fluorescence coincidence measurements based on close investigation of the coincidence correlation-matrix. This matrix contains temporal correlations between the number of detected bursts in individual channels and their coincidences. We show that the Euclidian norm of a vector Γ derived from elements of the correlation matrix takes a value between 0 and 2 depending on the relative coincidence frequency. We characterized the Γ-norm and its dependence on various experimental conditions by computer simulations and fluorescence microscopy experiments. Single-molecule experiments with two differently colored dye molecules diffusing freely in aqueous solution, a sample that generates purely random coincidence events, return a Γ-norm less than one, depending on the concentration of the fluorescent dyes. As perfect coincidence sample we monitored broad autofluorescence of 2.8 μm beads and determined the Γ-norm to be maximal and close to two. As in realistic diagnostic applications, we show that two-color coincidence detection of single-stranded DNA molecules, using differently labeled Molecular Beacons hybridizing to the same target, reveal a value between one and two representing a mixture of an optimal coincidence sample and a sample generating random coincidences. The Γ-norm introduced for data analysis provides a quantifiable measure for quickly judging the outcome of single-molecule coincidence experiments and estimating the quality of detected coincidences.     

Hydrodynamic manipulation of DNA in nanopost arrays: unhooking dynamics and size separation

Micro- or nanofabricated obstacle arrays are widely used as model matrices to perform fast DNA separations by electrophoresis. In this report, a gallery of obstacles of radii spanning from 40 to 250 nm are used to investigate the dynamics of hydrodynamic-field-driven DNA-nanopost collisions at the single-molecule level. The data shows that DNA disengagement dynamics are reasonably well described by conventional electrophoretic models in the limit of a large spacing between obstacles and for moderate migration velocities. It is also demonstrated that the use of hydrodynamic flow fields to convey DNA molecules is associated with changes in the configurational space of hooking events, and to altered relaxation dynamics between consecutive collisions. This study defines experimental conditions for the efficient separation of DNA fragments of tens of base pairs, and provides a complete framework by which to understand the behavior of DNA in the course of hydrodynamic-driven migrations through nanopost arrays.     

CECILIA,a versatile research tool for cellular responses to gravity

We describe a centrifuge designed and constructed according to current demands for a versatile instrument in cellular gravitational research, in particular protists (ciliates, flagellates). The instrument (called CECILIA,centrifuge forciliates) is suited for videomonitoring, videorecording, and quantitative evaluation of data from large numbers of swimming cells in a ground-based laboratory or in a drop tower/drop shaft under microgravity conditions. The horizontal rotating platform holds up to six 8mm-camcorders and six chambers holding the experimental cells. Under hypergravity conditions (up to 15 g) chambers can be rotated about 2 axes to adjust the swimming space at right angles or parallel to the resulting gravity vector. Evaluations of cellular responses to central acceleration — in the presence of gravitational 1 g — are used for extrapolation of cellular behaviour under hypogravity conditions. CECILIA is operated and monitored by computer using a custom-made soft-ware. Times and slopes of rising and decreasing acceleration, values and quality of steady acceleration are supervised online. CECILIA can serve as an on-ground research instrument for precursor investigations of the behaviour of ciliates and flagellates under microgravity conditions such as long-term experiments in the International Space Station.