Single Biological Macromolecules Research Articles

Particle trapping with optical nanofibers: a review [Invited

Optical trapping has proven to be an efficient method to control particles, including biological cells, single biological macromolecules, colloidal microparticles, and nanoparticles. Multiple types of particles have been successfully trapped, leading to various applications of optical tweezers ranging from biomedical through physics to material sciences. However, precise manipulation of particles with complex composition or of sizes down to nanometer-scales can be difficult with conventional optical tweezers, and an alternative manipulation tool is desirable. Optical nanofibers, that is, fibers with a waist diameter smaller than the propagating wavelength of light, are ideal candidates for optical manipulation due to their large evanescent field that extends beyond the fiber surface. They have the added advantages of being easily connected to a fibered experimental setup, being simple to fabricate, and providing strong electric field confinement and intense magnitude of evanescent fields at the nanofiber's surface. Many different particles have been trapped, rotated, transported, and assembled with such a system. This article reviews particle trapping using optical nanofibers and highlights some challenges and future potentials of this developing topic.

Effect of Coriolis and buoyancy forces on three-dimensional flow of chemically reactive tangent hyperbolic fluid subject to variable viscosity

Thermophoresis effect has wide range of applications in electro-static precipitators and in biology for calculating single biological macro molecules, such as genomic-length DNA and HIV virus in the micro channels. Current study deal with effects of Coriolis and buoyancy forces on the three-dimensional boundary layer flow of tangent hyperbolic fluid with thermo-migration and haphazard motion of nano-sized particles. Arrhenius kind of chemical reaction is taken along an exponentially stretchable surface. The main focus of current exploration is to execute shear thinning nano-liquid flow past an exponentially rotating stretchable surface under the influence of variable viscosity, mixed convection and activation energy. We are motivated to explore the features of three-dimensional shear thinning model combined under the features of mixed convention, variable viscosity, and activation energy. The mathematical model is designed to generate PDEs and converted them into ODEs by employing fractious transformation. The numerical outcomes are exhibited via graphs by employing Bvp4c numerical technique whereas the values of skin friction coefficient are calculated by monopolizing shooting method. Characteristics of the parameters appearing in modeling like the viscosity parameter, power-law index, local Weissenberg number, mix convection parameter, rotation parameter, Prandtl number and chemical reaction parameter are comprehensively analyzed through graphical behavior. The impact of governing parameters on skin friction, heat and mass transfer rates is illustrated through tables. The detail analysis anticipates that the elevation in Weissenberg number and porosity caused decline in velocity. Further, the temperature behaves doppositely analogous to development Prandtl besides the thermophoresis parameter.

Evanescent scattering imaging of single protein binding kinetics and DNA conformation changes

Evanescent illumination has been widely used to detect single biological macromolecules because it can notably enhance light-analyte interaction. However, the current evanescent single-molecule detection system usually requires specially designed microspheres or nanomaterials. Here we show that single protein detection and imaging can be realized on a plain glass surface by imaging the interference between the evanescent lights scattered by the single proteins and by the natural roughness of the cover glass. This allows us to quantify the sizes of single proteins, characterize the protein–antibody interactions at the single-molecule level, and analyze the heterogeneity of single protein binding behaviors. In addition, owing to the exponential distribution of evanescent field intensity, the evanescent imaging system can track the analyte axial movement with high resolution, which can be used to analyze the DNA conformation changes, providing one solution for detecting small molecules, such as microRNA. This work demonstrates a label-free single protein imaging method with ordinary consumables and may pave a road for detecting small biological molecules.

Noise reduction and mask removal neural network for X-ray single-particle imaging.

Free-electron lasers could enable X-ray imaging of single biological macromolecules and the study of protein dynamics, paving the way for a powerful new imaging tool in structural biology, but a low signal-to-noise ratio and missing regions in the detectors, colloquially termed 'masks', affect data collection and hamper real-time evaluation of experimental data. In this article, the challenges posed by noise and masks are tackled by introducing a neural network pipeline that aims to restore diffraction intensities. For training and testing of the model, a data set of diffraction patterns was simulated from 10 900 different proteins with molecular weights within the range of 10-100 kDa and collected at a photon energy of 8 keV. The method is compared with a simple low-pass filtering algorithm based on autocorrelation constraints. The results show an improvement in the mean-squared error of roughly two orders of magnitude in the presence of masks compared with the noisy data. The algorithm was also tested at increasing mask width, leading to the conclusion that demasking can achieve good results when the mask is smaller than half of the central speckle of the pattern. The results highlight the competitiveness of this model for data processing and the feasibility of restoring diffraction intensities from unknown structures in real time using deep learning methods. Finally, an example is shown of this preprocessing making orientation recovery more reliable, especially for data sets containing very few patterns, using the expansion-maximization-compression algorithm.

Ab initiocalculation of electron-impact-ionization cross sections for ions in exotic electron configurations

Atomic or molecular assemblies irradiated with intense hard x-ray pulses, such as those emitted from x-ray free-electron lasers (XFELs), are subject to a strong ionization, which also releases electrons from atomic inner shells. The resulting core-hole states relax via various channels, including fluorescence and Auger decay. The latter is the predominant relaxation channel for light elements and typically occurs on a time scale of 1--10 fs. In dense samples, the core-hole ions may already undergo electron-impact ionizations during this time due to the abundance of highly energetic photoelectrons and Auger electrons. In this study we perform an ab initio calculation of the electron-impact-ionization cross sections of ions with an arbitrary electronic configuration at zero temperature. This allows us to evaluate and compare impact-ionization cross sections for ions in ground and ``exotic'' electronic states (e.g., with a few core holes), which may be formed during their interaction with intense x-ray pulses. We show that the impact-ionization cross sections for ions of the same charge, but with varying electronic configurations, may significantly differ. This finding has to be taken into account in any modeling tool treating the relaxation of atoms after high-energy-impact collision, e.g., simulations dedicated for coherent x-ray diffraction imaging of nanocrystals and single biological macromolecules, or laser-created plasma studies. Our computationally efficient ab initio calculation scheme can be easily incorporated in such simulation schemes.

Quantitative mass imaging of single biological macromolecules.

The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.

Low-dose cryo electron ptychography via non-convex Bayesian optimization

Electron ptychography has seen a recent surge of interest for phase sensitive imaging at atomic or near-atomic resolution. However, applications are so far mainly limited to radiation-hard samples, because the required doses are too high for imaging biological samples at high resolution. We propose the use of non-convex Bayesian optimization to overcome this problem, and show via numerical simulations that the dose required for successful reconstruction can be reduced by two orders of magnitude compared to previous experiments. As an important application we suggest to use this method for imaging single biological macromolecules at cryogenic temperatures and demonstrate 2D single-particle reconstructions from simulated data with a resolution up to 5.4 Å at a dose of 20e−/Å2. When averaging over only 30 low-dose datasets, a 2D resolution around 3.5 Å is possible for macromolecular complexes even below 100 kDa. With its independence from the microscope transfer function, direct recovery of phase contrast, and better scaling of signal-to-noise ratio, low-dose cryo electron ptychography may become a promising alternative to Zernike phase-contrast microscopy.

Construction and calibration of an optical trap on a fluorescence optical microscope

The application of optical traps has come to the fore in the last three decades. They provide a powerful, sterile and noninvasive tool for the manipulation of cells, single biological macromolecules, colloidal microparticles and nanoparticles. An optically trapped microsphere may act as a force transducer that is used to measure forces in the piconewton regime. By setting up a well-calibrated single-beam optical trap within a fluorescence microscope system, one can measure forces and collect fluorescence signals upon biological systems simultaneously. In this protocol, we aim to provide a clear exposition of the methodology of assembling and operating a single-beam gradient force trap (optical tweezers) on an inverted fluorescence microscope. A step-by-step guide is given for alignment and operation, with discussion of common pitfalls.

Perspectives on Single Molecule Diffraction Using the X-Ray Free Electron Laser

A free electron laser capable of operating at the short wavelengths and high energies characteristic of hard X-rays, could be a potentially powerful new tool for the determination of the structures of single biological macromolecules, subcellular organelles, or whole cells at high resolution. Current methods using relatively low-intensity, incoherent X-ray sources depend upon the high signal gain achieved by the coherent scattering of a large number of identical molecules from a crystalline sample. The prerequisite crystallization of biological macromolecules is a major bottleneck in this process and the discrete nature of the Fourier transforms of crystalline materials also complicates the process of the reconstruction of the original molecular structure from its corresponding diffraction pattern. The ability to record detailed diffraction patterns from single molecules would eliminate the need for crystalline samples. Furthermore, the continuous molecular transforms of single molecules can be sampled finely enough to enable the unrecorded phase information to be retrieved from the set of structural amplitudes without recourse to the additional, a priori structural information that is generally required for phase assignment using sets of diffraction amplitudes from crystalline samples. Currently, diffraction experiments with single biological macromolecules or other non-crystalline material are hampered by the significant radiation damage to the samples that occurs as a result of the very large radiation doses that are required. Model calculations indicate that this problem may be solved by using the ultra-short (< 100 fs) pulses of hard X-radiation that a new generation of X-ray free electron lasers should be capable of producing. In anticipation that the current, rapid development of this technology will make this potential a reality in the near future, the theoretical foundations of this new methodology are already being laid.

Equations for Stochastic Macromolecular Mechanics of Single Proteins: Equilibrium Fluctuations, Transient Kinetics, and Nonequilibrium Steady-State

A modeling framework for the internal conformational dynamics and external mechanical movement of single biological macromolecules in aqueous solution at constant temperature is developed. Both the internal dynamics and the external movement are stochastic; the former is represented by a master equation for a set of discrete states, and the latter is described by a continuous Smoluchowski equation. Combining these two equations into one yields a comprehensive theory for the Brownian dynamics and statistical thermodynamics of single macromolecules. This approach is shown to have wide applications. It is applied to protein−ligand dissociation under external force, unfolding of polyglobular proteins under extension, movement along linear tracks of motor proteins against load, and enzyme catalysis by single fluctuating proteins. As a generalization of the classic polymer theory, the dynamic equation is capable of characterizing a single macromolecule in aqueous solution, in probabilistic terms, (1) its thermo...

Probing the Machinery of Intracellular Trafficking with the Atomic Force Microscope

Atomic force microscopy has emerged as a powerful tool for characterizing single biological macromolecules, macromolecular assemblies, and whole cells in aqueous buffer, in real time, and at molecular-scale spatial and force resolution. Many of the central elements of intracellular transport are tens to hundreds of nanometers in size and highly dynamic. Thus, atomic force microscopy provides a valuable means of addressing questions of structure and mechanism in intracellular transport. We begin this review of recent efforts to apply atomic force microscopy to problems in intracellular transport by discussing the technical principles behind atomic force microscopy. We then turn to three specific areas in which atomic force microscopy has been applied to problems with direct implications for intracellular trafficking: cytoskeletal structure and dynamics, vesicular transport, and receptor-ligand interactions. In each case, we discuss studies which use both intact cellular elements and reconstituted models. While many technical challenges remain, these studies point to several areas where atomic force microscopy can be used to provide valuable insight into intracellular transport at exquisite spatial and energetic resolution.

Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor.

We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.