Biophysical Applications Research Articles

Plasmonic-based impedance microspectroscopy of optically heterogeneous samples.

A robust impedance microscopy technique is presented. This optical tool enables high resolution imaging of electrical properties with promising biophysical applications. The underlying principle is that surface plasmon resonance (SPR) sensors are able to measure perturbations of surface charge density and therefore can be used to compute the impedance of surface-adhered cells. However, the ability to perform reliable quantitative impedance imaging is affected by the optical heterogeneity of the cell-sensor interface. To address this issue, a novel method for quantitative time-resolved resonance angle tracking is developed and applied to correct for the effect of the optical properties. To demonstrate the capability of this technique, impedance microspectroscopy of bovine serum albumin (BSA) patterns was performed enabling measurements of capacitance with submicroscopic resolution. The work presented offers an impedance microspectroscopy method that will create new avenues in studying the electrical properties of single cells and biomolecules as well as bio-electrical currents.

Single-Chain Lanthanide Luminescence Biosensors for Cell-Based Imaging and Screening of Protein-Protein Interactions.

SummaryLanthanide-based, Förster resonance energy transfer (LRET) biosensors enabled sensitive, time-gated luminescence (TGL) imaging or multiwell plate analysis of protein-protein interactions (PPIs) in living cells. We prepared stable cell lines that expressed polypeptides composed of an alpha helical linker flanked by a Tb(III) complex-binding domain, GFP, and two interacting domains at each terminus. The PPIs examined included those between FKBP12 and the rapamycin-binding domain of m-Tor (FRB) and between p53 (1–92) and HDM2 (1–128). TGL microscopy revealed dramatic differences (>500%) in donor- or acceptor-denominated, Tb(III)-to-GFP LRET ratios between open (unbound) and closed (bound) states of the biosensors. We observed much larger signal changes (>2,500%) and Z′-factors of 0.5 or more when we grew cells in 96- or 384-well plates and analyzed PPI changes using a TGL plate reader. The modular design and exceptional dynamic range of lanthanide-based LRET biosensors will facilitate versatile imaging and cell-based screening of PPIs.

Metal Nanoapertures and Single Emitters

AbstractNanoapertures in metal films have long been recognized for their ability to confine light to subwavelength dimensions. This allows for high‐resolution imaging of single emitters with enhanced emission rates. It also provides a background‐free environment that has been exploited for biophysical applications like fluorescence correlation studies at high physiological concentrations and DNA sequencing. Recently, other advantages of single apertures have been recognized, including optical trapping, thermal management, and nanofluidic (nanopore) functionality. This paper reviews metal nanoapertures and their applications to single emitter studies for biophysical and quantum applications.

Vascularization Strategies in the Prevention of Nonunion Formation.

Delayed healing and nonunion formation are major challenges in orthopedic surgery, which require the development of novel treatment strategies. Vascularization is considered one of the major prerequisites for successful bone healing, providing an adequate nutrient supply and allowing the infiltration of progenitor cells to the fracture site. Hence, during the last decade, a considerable number of studies have focused on the evaluation of vascularization strategies to prevent or to treat nonunion formation. These involve (1) biophysical applications, (2) systemic pharmacological interventions, and (3) tissue engineering, including sophisticated scaffold materials, local growth factor delivery systems, cell-based techniques, and surgical vascularization approaches. Accumulating evidence indicates that in nonunions, these strategies are indeed capable of improving the process of bone healing. The major challenge for the future will now be the translation of these strategies into clinical practice to make them accessible for the majority of patients. If this succeeds, these vascularization strategies may markedly reduce the incidence of nonunion formation. Impact statement Delayed healing and nonunion formation are a major clinical problem in orthopedic surgery. This review provides an overview of vascularization strategies for the prevention and treatment of nonunions. The successful translation of these strategies in clinical practice is of major importance to achieve adequate bone healing.

Flexoelectric fluid membrane vesicles in spherical confinement

The morphology of spherically confined flexoelectric fluid membrane vesicles in an external uniform electric field is studied numerically. Due to the deformations induced by the confinement, the membrane becomes polarized resulting in an interaction with the external field. The equilibrium shapes of the vesicle without electric field can be classified in a geometrical phase diagram as a function of scaled area and reduced volume (Kahraman O. et al., EPL, 97 (2012) 68008; Kahraman O. et al., New. J. Phys., 14 (2012) 095021). When the area of the membrane is only slightly larger than the area of the confining sphere, a single axisymmetric invagination appears. A non-vanishing electric field induces an additional elongation of the confined vesicle which is either perpendicular or parallel depending on the sign of the electric field parameter. Higher values of the surface area or the electric field parameter can reduce the symmetry of the system leading to more complex folding. We present the resulting shapes and show that transition lines are shifted in the presence of an electric field. The obtained folding patterns could be of interest for biophysical and technological applications alike.

Diffusion toward non-overlapping partially reactive spherical traps: Fresh insights onto classic problems.

Several classic problems for particles diffusing outside an arbitrary configuration of non-overlapping partially reactive spherical traps in three dimensions are revisited. For this purpose, we describe the generalized method of separation of variables for solving boundary value problems of the associated modified Helmholtz equation. In particular, we derive a semi-analytical solution for the Green function that is the key ingredient to determine various diffusion-reaction characteristics such as the survival probability, the first-passage time distribution, and the reaction rate. We also present modifications of the method to determine numerically or asymptotically the eigenvalues and eigenfunctions of the Laplace operator and the Dirichlet-to-Neumann operator in such perforated domains. Some potential applications in chemical physics and biophysics are discussed, including diffusion-controlled reactions for mortal particles.

Fragment Quantum Mechanical Method for Excited States of Proteins: Development and Application to the Green Fluorescent Protein.

Understanding the excited-state properties of luminescent biomolecules is of central importance to their biophysical applications. In this study, we develop the Electrostatically Embedded Generalized Molecular Fractionation with Conjugate Caps (EE-GMFCC) method for quantitatively characterizing properties of covalently bonded systems with localized excitations (i.e., involving a single chromophore), such as fluorescent proteins. The excitation energy, transition dipole moment, and oscillator strength of wild-type Green Fluorescent Protein (wt-GFP) calculated by EE-GMFCC are found to be in excellent agreement with full system time-dependent density functional theory results. We also applied the Polarized Protein-Specific Charge model to wt-GFP, and found that electronic polarization of the protein is critical in stabilizing hydrogen bonding interactions in wt-GFP, which influences its absorption spectrum. The predicted absorption spectra of wt-GFP in the A and B states qualitatively agree with experiment. The fragmentation approach further allows a straightforward per residue decomposition of the excitation which reveals the influence of the protein environment on the absorption spectra of wt-GFP A and B states. Our results demonstrate that the EE-GMFCC method is both accurate and efficient for excited-state property calculations on proteins.

Entrapment of calf-thymus DNA on magnetic nanoparticles

Magnetic nanoparticles can be used to load bio–molecules for various biophysical applications. The direct attachment of bio–molecules such as protein and DNA to magnetic nanoparticles may lead to alter their structure. A method to immobilize and store biomolecules for example DNA on to a magnetic nanoparticles surface without much structural alteration is carried out in the present work. DNA is a target for many therapeutic small molecules as therapeutic drugs bind to DNA – interfering with protein factors involved in DNA mechanism, or cleave DNA cross-link – interfering with the cell division. This may find applications in drug interactions study with the stored DNA without much alteration in their structure.

Quinoline Containing Side-chain Antimalarial Analogs: Recent Advances and Therapeutic Application.

The side-chains of quinoline antimalarial agents are the major concern of focus to build novel and efficaciaous bioactive and clinical antimalarials. Bioative antimalarial analogs may play a critical role in pH trapping in the food vacuole of RBC's with the help of fragmented amino acid, thus lead to β-hematin inhibition. Here, the authors tried to summarize a useful, comprehensive compilation of side-chain modified ACQs along with their synthesis, biophysical and therapeutic applications etc. of potent antiplasmodial agents and therefore, opening the door towards the potential clinical status.

Applied connectivity modelling at local to regional scale: The potential for sea lice transmission between Scottish finfish aquaculture management areas

Due to the importance of parasite and disease controls for animal welfare, environmental sustainability and the economic viability of the finfish aquaculture industry in coastal waters, spatial management measures need to take into account the degree of connectivity between management areas. This paper describes the model integration (one-way nesting) of local high-resolution and wider domain hydrodynamic models around Scotland and the combination of their outputs to force a bio-physical modelling application to support marine spatial management. We used climatological flow fields from a hydrodynamic modelling framework (the “Scottish Shelf Model”) to estimate the degree of connectivity between Scottish finfish aquaculture Farm Management Areas (FMA) using bio-physical simulations of virtual organisms representing the planktonic phase of parasitic sea lice, an important problem for the aquaculture industry. Three types of analysis are carried out: presence/absence of connections between FMAs as the most extreme case, connection probabilities above a defined biologically-meaningful threshold, and above-threshold connection probabilities with a weighting associated with farmed fish within-area biomass (as a proxy for sea lice production) as a more realistic scenario. We find a general northward flow of particles from mainland farms aligned with the prevailing circulation and high connection probability towards the north. Some regions (Loch Linnhe/Sound of Mull) can be classified as net exporters and some (east coast of the Western Isles, the north-west coast) as net receivers of particles. A few selected regions show far reaching connections above the biologically-meaningful probability threshold, for example across the Minch between the Scottish mainland and the Western Isles. Other areas such as Shetland appear to be self-contained and internally well inter-connected. Our results are relevant to the marine aquaculture industry and regulators as they can provide useful information that can be used to develop effective strategies for parasite control.

Multiplexed Nanometric 3D Tracking of Microbeads Using an FFT-Phasor Algorithm

Many single-molecule biophysical techniques rely on nanometric tracking of microbeads to obtain quantitative information about the mechanical properties of biomolecules such as chromatin fibers. Their three-dimensional (3D) position can be resolved by holographic analysis of the diffraction pattern in wide-field imaging. Fitting this diffraction pattern to Lorenz-Mie scattering theory yields the bead’s position with nanometer accuracy in three dimensions but is computationally expensive. Real-time multiplexed bead tracking therefore requires a more efficient tracking method, such as comparison with previously measured diffraction patterns, known as look-up tables. Here, we introduce an alternative 3D phasor algorithm that provides robust bead tracking with nanometric localization accuracy in a z range of over 10 μm under nonoptimal imaging conditions. The algorithm is based on a two-dimensional cross correlation using fast Fourier transforms with computer-generated reference images, yielding a processing rate of up to 10,000 regions of interest per second. We implemented the technique in magnetic tweezers and tracked the 3D position of over 100 beads in real time on a generic CPU. The accuracy of 3D phasor tracking was extensively tested and compared to a look-up table approach using Lorenz-Mie simulations, avoiding experimental uncertainties. Its easy implementation, efficiency, and robustness can improve multiplexed biophysical bead-tracking applications, especially when high throughput is required and image artifacts are difficult to avoid.

Ferromagnetic Microwire Systems as a High-Gradient Magnetic Field Source for Magnetophoresis

The use of amorphous ferromagnetic microwires has potential for applications in biomedicine and biophysics. Microwire arrays create magnetic fields that are characterized by strong spatial gradients. A system of ferromagnetic microwires with magnetizations across the diameter may efficiently accumulate paramagnetic particles along the wire due to forced diffusion. For typical system parameters, the particles concentration at the wire surface increases more than twice for a characteristic time of 30 s. A pair of closely spaced microwires with diametric magnetizations (referred to as a dipole pair) yields a unique potential landscape with a two-dimensional minimum. The possibility of levitation of diamagnetic particles just above the dipole pair array was numerically demonstrated.

Functionalization of graphene quantum dots (GQDs) with chitosan biopolymer for biophysical applications

Graphene quantum dots (GQDs) have become one of the potential materials in several applications. Recently, functionalization of GQDs with various functional groups is one of the hot spots of research. Chitosan (CS) biopolymer is used in functionalizing GQDs in order to enhance its mechanical properties. Molecular modeling with different levels including molecular dynamics; Quantum Mechanics as well Quantitative Structure Activity Relationship are widely applied to study emerging materials such as GQDs. As a computational case study, two interaction probabilities are proposed. Physical interaction mechanism through OH and NH2 of CS is most probable to occur. Feasibility of utilizing the functionalized GQD is studied in interacting with ibuprofen. The resultant structure seems to be stable and highly reactive. This ensures the feasibility of the usage of GQDs in biological applications after some modifications.

Generalized Langevin Equation: An Introductory Review for Biophysicists

An introductory, pedagogical review of the generalized Langevin equation (GLE) within the classical regime is presented. It is intended to be accessible to biophysicists with an interest in molecular dynamics (MD). Section 1 presents why the equation may be of interest within biophysical modeling. A detailed elementary first principles derivation of the (multidimensional) Kac–Zwanzig model is presented. The literature is reviewed with a focus on biophysical applications and representation by Markovian stochastic differential equations. The relationship with the Mori–Zwanzig formalism is discussed. The framework of model reduction and approximation is emphasized. Some open problems are identified.

Photophysical properties of the hemicyanine Dy-630 and its potential as a single-molecule fluorescent probe for biophysical applications

Protein-induced fluorescence enhancement (PIFE) is an increasingly used approach to investigate DNA-protein interactions at the single molecule level. The optimal probe for this type of application is highly photostable, has a high absorption extinction coefficient, and has a moderate fluorescence quantum yield that increases significantly when the dye is in close proximity to a large macromolecule such as a protein. So far, the green-absorbing symmetric cyanine known as Cy3 has been the probe of choice in this field because the magnitude of the increase observed upon protein binding (usually 2–4 –fold) is large enough to allow for the analysis of protein dynamics on the inherently noisy single-molecule signals. Here, we report the characterization of the photophysical properties of the red-absorbing hemicyanine dye Dy-630 in the context of its potential application as a single-molecule PIFE probe. The behavior of Dy-630 in solution is similar to that of Cy3; the fluorescence quantum yield and lifetime of Dy-630 increase with increasing viscosity, and decrease with increasing temperature indicating the existence of an activated nonradiative process that depopulates the singlet state of the dye. As in the case of Cy3, the results of transient spectroscopy experiments are consistent with the formation of a photoisomer that reverts to the ground state thermally in the microsecond timescale. Unfortunately, experiments with DNA samples paint a more complex scenario. As in the case of Cy3, the fluorescence quantum yield of Dy-630 increases significantly when the dye interacts with the DNA bases, but in the case of Dy-630 attachment to DNA results in an already long fluorescence lifetime that does not provide a significant window for the protein-induced enhancement observed with Cy3. Although we show that Dy-630 may not be well-suited for PIFE, our results shed light on the optimal design principles for probes for PIFE applications.

Robotic platform for microinjection into single cells in brain tissue

Microinjection into single cells in brain tissue is a powerful technique to study and manipulate neural stem cells. However, such microinjection requires expertise and is a low‐throughput process. We developed the “Autoinjector”, a robot that utilizes images from a microscope to guide a microinjection needle into tissue to deliver femtoliter volumes of liquids into single cells. The Autoinjector enables microinjection of hundreds of cells within a single organotypic slice, resulting in an overall yield that is an order of magnitude greater than manual microinjection. The Autoinjector successfully targets both apical progenitors (APs) and newborn neurons in the embryonic mouse and human fetal telencephalon. We used the Autoinjector to systematically study gap‐junctional communication between neural progenitors in the embryonic mouse telencephalon and found that apical contact is a characteristic feature of the cells that are part of a gap junction‐coupled cluster. The throughput and versatility of the Autoinjector will render microinjection an accessible high‐performance single‐cell manipulation technique and will provide a powerful new platform for performing single‐cell analyses in tissue for bioengineering and biophysics applications.

Nanoscale Mapping of Nonuniform Heterogeneous Nucleation Kinetics Mediated by Surface Chemistry.

Nucleation underlies the formation of many liquid-phase synthetic and natural materials with applications in materials chemistry, geochemistry, biophysics, and structural biology. Most liquid-phase nucleation processes are heterogeneous, occurring at specific nucleation sites at a solid-liquid interface; however, the chemical and topographical identity of these nucleation sites and how nucleation kinetics vary from site-to-site remain mysterious. Here we utilize in situ liquid cell electron microscopy to unveil counterintuitive nanoscale nonuniformities in heterogeneous nucleation kinetics on a macroscopically uniform solid-liquid interface. Time-resolved in situ electron microscopy imaging of silver nanoparticle nucleation at a water-silicon nitride interface showed apparently randomly located nucleation events at the interface. However, nanometric maps of local nucleation kinetics uncovered nanoscale interfacial domains with either slow or rapid nucleation. Interestingly, the interfacial domains vanished at high supersaturation ratio, giving way to rapid spatially uniform nucleation kinetics. Atomic force microscopy and nanoparticle labeling experiments revealed a topographically flat, chemically heterogeneous interface with nanoscale interfacial domains of functional groups similar in size to those observed in the nanometric nucleation maps. These results, along with a semiquantitative nucleation model, indicate that a chemically nonuniform interface presenting different free energy barriers to heterogeneous nucleation underlies our observations of nonuniform nucleation kinetics. Overall, our results introduce a new imaging modality, nanometric nucleation mapping, and provide important new insights into the impact of surface chemistry on microscopic spatial variations in heterogeneous nucleation kinetics that have not been previously observed.

Nanopore-based sensing interface for single molecule electrochemistry

Nanopore techniques are experiencing a gallop since it walked out the notebook and show its charm on the science arena. The nanoscale pore offers a single-molecule resolution with a label-free and high-selective manner for the research of molecular structures, molecular dynamics, single-molecule reactions and for a variety of applications in biophysics and bionanotechnology. In this review, we introduce the construction of three types of nanopore platforms along with the latest progress in DNA sensing, structure and dynamics analysis of peptides/proteins, and the detection of redox reactions with new sensing mechanisms. Then, we depict nanopore data processing methods which provide an insight of data mining under the background of big data. We could fully expect the great impact of nanopore techniques on not only for DNA sequencing and sensing applications, but also in protein sequencing and clinical diagnostics.

Perturbation of Transmission Matrices in Nonlinear Random Media

AbstractRandom media with tailored optical properties are attracting burgeoning interest for applications in imaging, biophysics, energy, nanomedicine, spectroscopy, cryptography, and telecommunications. A key paradigm for devices based on this class of materials is the transmission matrix, the tensorial link between the input and the output signals, that describes in full their optical behavior. The transmission matrix has specific statistical properties, such as the existence of lossless channels, that can be used to transmit information, and are determined by the disorder distribution. In nonlinear materials, these channels may be modulated and the transmission matrix tuned accordingly. Here, the direct measurement of the nonlinear transmission matrix of complex materials is reported, exploiting the strong optothermal nonlinearity of scattering silica aerogel (SA). It is shown that the dephasing effects due to nonlinearity are both controllable and reversible, opening the road to applications based on the nonlinear response of random media.

Controlled micelle conjugation via charged peptide amphiphiles.

We report the first demonstration of nonionic detergent micelle conjugation and phase separation using purpose-synthesized, peptide amphiphiles, C10 -(Asp)5 and C10 -(Lys)5 . Clustering is achieved in two different ways. Micelles containing the negatively charged peptide amphiphile C10 -(Asp)5 are conjugated (a) via a water-soluble, penta-Lys mediator or (b) to micelles containing the C10 -(Lys)5 peptide amphiphile. Both routes lead to phase separation in the form of oil-rich globules visible in the light microscope. The hydrophobic nature of these regions leads to spontaneous partitioning of hydrophobic dyes into globules that were found to be stable for weeks to months. Extension of the conjugation mechanism to micelles containing a recently discovered, light-driven proton pump King Sejong 1-2 (KS1-2) demonstrates that a membrane protein may be concentrated using peptide amphiphiles while preserving its native conformation as determined by characteristic UV absorption. The potential utility of these peptide amphiphiles for biophysical and biomedical applications is discussed.