Long-range formation of the Bicoid gradient requires multiple dynamic modes that spatially vary across the embryo

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

Measuring sub-nanometer undulations at microsecond temporal resolution with metal- and graphene-induced energy transfer spectroscopy

Abstract Out-of-plane fluctuations, also known as stochastic displacements, of biological membranes play a crucial role in regulating many essential life processes within cells and organelles. Despite the availability of various methods for quantifying membrane dynamics, accurately quantifying complex membrane systems with rapid and tiny fluctuations, such as mitochondria, remains a challenge. In this work, we present a methodology that combines metal/graphene-induced energy transfer (MIET/GIET) with fluorescence correlation spectroscopy (FCS) to quantify out-of-plane fluctuations of membranes with simultaneous spatiotemporal resolution of approximately one nanometer and one microsecond. To validate the technique and spatiotemporal resolution, we measure bending undulations of model membranes. Furthermore, we demonstrate the versatility and applicability of MIET/GIET-FCS for studying diverse membrane systems, including the widely studied fluctuating membrane system of human red blood cells, as well as two unexplored membrane systems with tiny fluctuations, a pore-spanning membrane, and mitochondrial inner/outer membranes

Metal-induced energy transfer

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

Excitation and Emission Transition Dipoles of Type-II Semiconductor Nanorods.

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

Quantifying Microsecond Transition Times Using Fluorescence Lifetime Correlation Spectroscopy

Many complex luminescent emitters such as fluorescent proteins exhibit multiple emitting states that result in rapid fluctuations of their excited-state lifetime. Here, we apply fluorescence lifetime correlation spectroscopy (FLCS) to resolve the photophysical state dynamics of the prototypical fluorescence protein enhanced green fluorescent protein (EGFP). We quantify the microsecond transition rates between its two fluorescent states, which have otherwise highly overlapping emission spectra. We relate these transitions to a room-temperature angstrom-scale rotational isomerism of an amino acid next to its fluorescent center. With this study, we demonstrate the power of FLCS for studying the rapid transition dynamics of a broad range of light-emitting systems with complex multistate photophysics, which cannot be easily done by other methods

Charge-Driven Fluorescence Blinking in Carbon Nanodots

This study focuses on the mechanism of fluorescence blinking of single carbon nanodots, which is one of their key but less understood properties. The results of our single-particle fluorescence study show that the mechanism of carbon nanodots blinking has remarkable similarities with that of semiconductor quantum dots. In particular, the temporal behavior of carbon nanodot blinking follows a power law both at room and at cryogenic temperatures. Our experimental data suggest that static quenching via Dexter-type electron transfer between surface groups of a nanoparticle plays a major role in the transition of carbon nanodots to off or gray states, whereas the transition back to on states is governed by an electron tunneling from the particle’s core. These findings advance our understanding of the complex mechanism of carbon nanodots emission, which is one of the key steps for their application in fluorescence imaging