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The mechanisms of exciton generation and recombination in semiconductor nanocrystals are crucial to the understanding of their photophysics and for their application in nearly all fields. While many studies have been focused on type-I heterojunction nanocrystals, the photophysics of type-II nanorods, where the hole is located in the core and the electron is located in the shell of the nanorod, remain largely unexplored. In this work, by scanning single nanorods through the focal spot of radially and azimuthally polarized laser beams and by comparing the measured excitation patterns with a theoretical model, we determine the dimensionality of the excitation transition dipole of single type-II nanorods. Additionally, by recording defocused patterns of the emission of the same particles, we measure their emission transition dipoles. The combination of these techniques allows us to unambiguously deduce the dimensionality and orientation of both excitation and emission transition dipoles of single type-II semiconductor nanorods. The results show that in contrast to previously studied quantum emitters, the particles possess a 3D degenerate excitation and a fixed linear emission transition dipole.

Cells crucially rely on the interactions of biomolecules at their plasma membrane to maintain homeostasis. Yet, a methodology to systematically quantify biomolecular organisation, measuring diffusion dynamics and oligomerisation, represents an unmet need. Here, we introduce the brightness-transit statistics (BTS) method based on fluorescence fluctuation spectroscopy and combine information from brightness and transit times to elucidate biomolecular diffusion and oligomerisation in both cell-free in vitro and in vitro systems incorporating living cells. We validate our approach in silico with computer simulations and experimentally using oligomerisation of EGFP tethered to supported lipid bilayers. We apply our pipeline to study the oligomerisation of CD40 ectodomain in vitro and endogenous CD40 on primary B cells. While we find a potential for CD40 to oligomerize in a concentration or ligand depended manner, we do not observe mobile oligomers on B cells. The BTS method combines sensitive analysis, quantification, and intuitive visualisation of dynamic biomolecular organisation. Methods to assess diffusion dynamics and oligomerisation of biomolecules remain scarce. Here, the authors present a brightness-transit statistics method to measure diffusion and oligomerisation simultaneously with high sensitivity, and they apply their approach to study the organisation of CD40. [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]

Since about a decade, metal-induced energy transfer (MIET) has become a tool to measure the distance of fluorophores to a metal-coated surface with nanometer accuracy. The energy transfer from a fluorescent molecule to surface plasmons within a metal film results in the acceleration of its radiative decay rate. This can be observed as a reduction of the molecule’s fluorescence lifetime which can be easily measured with standard microscopy equipment. The achievable distance resolution is in the nanometer range, over a total range of about 200 nm. The method is perfectly compatible with biological and even live cell samples. In this review, we will summarize the theoretical and technical details of the method and present the most important results that have been obtained using MIET. We will also show how the latest technical developments can contribute to improving MIET, and we sketch some interesting directions for its future applications in the life sciences.

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]

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]

Coupling of Atg8 to phosphatidylethanolamine is crucial for the expansion of the crescent-shaped phagophore during cargo engulfment. Atg21, a PtdIns3P-binding beta-propeller protein, scaffolds Atg8 and its E3-like complex Atg12–Atg5-Atg16 during lipidation. The crystal structure of Atg21, in complex with the Atg16 coiled-coil domain, showed its binding at the bottom side of the Atg21 beta-propeller. Our structure allowed detailed analyses of the complex formation of Atg21 with Atg16 and uncovered the orientation of the Atg16 coiled-coil domain with respect to the membrane. We further found that Atg21 was restricted to the phagophore edge, near the vacuole, known as the vacuole isolation membrane contact site (VICS). We identified a specialized vacuolar subdomain at the VICS, typical of organellar contact sites, where the membrane protein Vph1 was excluded, while Vac8 was concentrated. Furthermore, Vac8 was required for VICS formation. Our results support a specialized organellar contact involved in controlling phagophore elongation. Abbreviations: FCCS: fluorescence cross correlation spectroscopy; NVJ: nucleus-vacuole junction; PAS: phagophore assembly site; PE: phosphatidylethanolamine; PROPPIN: beta-propeller that binds phosphoinositides; PtdIns3P: phosphatidylinositol- 3-phosphate; VICS: vacuole isolation membrane contact site. [ABSTRACT FROM AUTHOR]