Search

Our paper presents the first theoretical and experimental study using single-molecule Metal-Induced Energy Transfer (smMIET) for localizing single fluorescent molecules in three dimensions. Metal-Induced Energy Transfer describes the resonant energy transfer from the excited state of a fluorescent emitter to surface plasmons in a metal nanostructure. This energy transfer is strongly distance-dependent and can be used to localize an emitter along one dimension. We have used Metal-Induced Energy Transfer in the past for localizing fluorescent emitters with nanometer accuracy along the optical axis of a microscope. The combination of smMIET with single-molecule localization based super-resolution microscopy that provides nanometer lateral localization accuracy offers the prospect of achieving isotropic nanometer localization accuracy in all three spatial dimensions. We give a thorough theoretical explanation and analysis of smMIET, describe its experimental requirements, also in its combination with lateral single-molecule localization techniques, and present first proof-of-principle experiments using dye molecules immobilized on top of a silica spacer, and of dye molecules embedded in thin polymer films. [ABSTRACT FROM AUTHOR]

To date, we could not engineer Nature's ability to dynamically handle diffusing single molecules in the liquid-phase as it takes place in pore-forming proteins and tunnelling nanotubes. Consistent handling of individual single molecules in a liquid is of paramount importance to fundamental molecular studies and technological benefits, like single-molecule level separation and sorting for early biomedical diagnostics, microscopic studies of molecular interactions and electron/optical microscopy of molecules and nanomaterials. We can consistently resolve the dynamics of diffusing single molecules if they are confined within a uniform dielectric environment at nanometre length-scales. A uniform dielectric environment is the key characteristic since intrinsic electronic properties of molecules were modified while interacting with any surfaces, and the effect is not the same from one dielectric surface to another. We present dynamic nanofluidic detection of optically active single molecules in a liquid. An all-silica nanofluidic environment was used to electrokinetically handle individual single-molecules where molecular shot noise was resolved. We recorded the single-molecule motion of small fragments of DNA, carbon-nanodots, and organic fluorophores in water. The electrokinetic 1D molecular mass transport under two-focus fluorescence correlation spectroscopy (2fFCS) showed confinement-induced modified molecular interactions (due to various inter-molecular repulsive and attractive forces), which have been theoretically interpreted as molecular shot noise. Our demonstration of high-throughput nanochannel fabrication, 2fFCS-based 1D confined detection of fast-moving single molecules and fundamental understanding of molecular shot noise may open an avenue for single-molecule experiments where physical manipulation of dynamics is necessary. [ABSTRACT FROM AUTHOR]

We describe an exact and highly efficient numerical algorithm for solving a special but important class of convection-diffusion equations. These equations occur in many problems in physics, chemistry, or biology, and they are usually hard to treat due to the presence of a convection term, represented by a first order derivative in the spatial co-ordinate. Our algorithm reduces the convection-diffusion equation to a pure diffusion problem within a complex-valued potential which can be solved efficiently and accurately using conventional parabolic partial differential equation solvers. [ABSTRACT FROM AUTHOR]

This chapter presents a concise introduction into the method of Fluorescence Lifetime Correlation Spectroscopy (FLCS). This is an extension of Fluorescence Correlation Spectroscopy (FCS) that analyses fluorescence intensity fluctuations from small detection volumes in samples of ultra-low concentration. FCS has been widely used for investigating diffusion, conformational changes, molecular binding/unbinding equilibria, or chemical reaction kinetics, at single molecule sensitivity. In FCS, this is done by calculating intensity correlation curves for the measured intensity fluctuations. FLCS extends this idea by calculating fluorescence-lifetime specific intensity correlation curves. Thus, FLCS is the method of choice for all studies where a parameter of interest (conformational state, spatial position, molecular environmental condition) is connected with a change in the fluorescence lifetime. After presenting the theoretical and experimental basis of FLCS, the chapter gives an overview of its various applications. [ABSTRACT FROM AUTHOR]

Single-molecule localization based super-resolution microscopy has revolutionized optical microscopy and routinely allows for resolving structural details down to a few nanometers. However, there exists a rather large discrepancy between lateral and axial localization accuracy, the latter typically three to five times worse than the former. Here, we use single-molecule metal-induced energy transfer (smMIET) to localize single molecules along the optical axis, and to measure their axial distance with an accuracy of 5 nm. smMIET relies only on fluorescence lifetime measurements and does not require additional complex optical setups. [ABSTRACT FROM AUTHOR]

The emission properties of most fluorescent emitters, such as dye molecules or solid-state color centers, can be well described by the model of an oscillating electric dipole. However, the orientations of their excitation and emission dipoles are, in most cases, not parallel. Although single molecule excitation and emission dipole orientation measurements have been performed in the past, no experimental method has so far looked at the three-dimensional excitation and emission dipole geometry of individual emitters simultaneously. We present the first experimental study, using defocused imaging in conjunction with radially polarized excitation scanning, to measure both the excitation as well as emission dipole orientations of single molecules, which allows us to sample the distribution of their mutual orientation. We find an unexpectedly broad distribution of the angle between both dipoles which we attribute to the interaction between the observed molecules and the substrate they are immobilized on. [ABSTRACT FROM AUTHOR]

From molecules and particles to macroscopic surfaces immersed in fluids, chemical reactions often endow interfaces with electrical charge which in turn governs surface interactions and interfacial phenomena. The ability to measure the electrical properties of a material immersed in any solvent, as well as to monitor the spatial heterogeneity and temporal variation thereof, has been a long-standing challenge. Here, we describe an optical microscopy-based approach to probe the surface charge distribution of a range of materials, including inorganic oxide, polymer, and polyelectrolyte films, in contact with a fluid. The method relies on optical visualization of the electrical repulsion between diffusing charged probe molecules and the unknown surface to be characterized. Rapid image-based measurements enable us to further determine isoelectric points of the material as well as properties of its ionizable chemical groups. We further demonstrate the ability to optically monitor chemically triggered surface charge changes with millisecond time resolution. Finally, we present a scanning-surface probe technique capable of diffraction-limited imaging of spatial heterogeneities in chemical composition and charge over large areas. This technique will enable facile characterization of the solid-liquid interface with wide-ranging relevance across application areas from biology to engineering. [ABSTRACT FROM AUTHOR]

Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range. [ABSTRACT FROM AUTHOR]

A large number of transport processes in physics, chemistry, and engineering are described by a convection–diffusion equation. This equation is notoriously difficult to solve due to the presence of convection-related first-order gradient differential operators. We describe a new and efficient numerical method for solving the convection–diffusion equation for laminar flows within channels of arbitrary cross section. It is based on reducing the convection–diffusion equation to a set of pure diffusion equations with a complex-valued potential for which fast and numerically stable solvers are readily available. Additionally, we use an eigenvector projection method that allows us to compute snapshots of full concentration distributions over millions of finite elements within a few seconds using a conventional state-of-the-art desktop computer. Our results will be important for all applications where diffusion and convection are both important for correctly describing material transport [ABSTRACT FROM AUTHOR]

We model the transport of electrically charged solute molecules by a laminar flow within a nanoslit microfluidic channel with electrostatic surface potential. We derive the governing convection–diffusion equation, solve it numerically, and compare it with a Taylor–Aris-like approximation, which gives excellent results for small Péclet numbers. We discuss our results in light of designing an assay that can measure simultaneously the hydrodynamic size and electric charge of single molecules by tracking their motion in such nanoslit channels with electrostatic surface potential. [ABSTRACT FROM AUTHOR]

Quantifying molecular dynamics within the context of complex cellular morphologies is essential toward understanding the inner workings and function of cells. Fluorescence recovery after photobleaching (FRAP) is one of the most broadly applied techniques to measure the reaction diffusion dynamics of molecules in living cells. FRAP measurements typically restrict themselves to single‐plane image acquisition within a subcellular‐sized region of interest due to the limited temporal resolution and undesirable photobleaching induced by 3D fluorescence confocal or widefield microscopy. Here, an experimental and computational pipeline combining lattice light sheet microscopy, FRAP, and numerical simulations, offering rapid and minimally invasive quantification of molecular dynamics with respect to 3D cell morphology is presented. Having the opportunity to accurately measure and interpret the dynamics of molecules in 3D with respect to cell morphology has the potential to reveal unprecedented insights into the function of living cells. [ABSTRACT FROM AUTHOR]

The biological process of the epithelial-to-mesenchymal transition (EMT) allows epithelial cells to enhance their migratory and invasive behavior and plays a key role in embryogenesis, fibrosis, wound healing, and metastasis. Among the multiple biochemical changes from an epithelial to a mesenchymal phenotype, the alteration of cellular dynamics in cell-cell as well as cell-substrate contacts is crucial. To determine these variations over the whole time scale of the EMT, we measure the cell-substrate distance of epithelial NMuMG cells during EMT using our newly established metal-induced energy transfer (MIET) microscopy, which allows one to achieve nanometer axial resolution. We show that, in the very first hours of the transition, the cell-substrate distance increases substantially, but later in the process after reaching the mesenchymal state, this distance is reduced again to the level of untreated cells. These findings relate to a change in the number of adhesion points and will help to better understand remodeling processes associated with wound healing, embryonic development, cancer progression, or tissue regeneration. [ABSTRACT FROM AUTHOR]