Tissue multicolor STED nanoscopy of presynaptic proteins in the calyx of Held.

The calyx of Held, a large glutamatergic terminal in the mammalian auditory brainstem has been extensively employed to study presynaptic structure and function in the central nervous system. Nevertheless, the nanoarchitecture of presynaptic proteins and subcellular components in the calyx terminal and its relation to functional properties of synaptic transmission is only poorly understood. Here, we use stimulated emission depletion (STED) nanoscopy of calyces in thin sections of aldehyde-fixed rat brain tissue to visualize immuno-labeled synaptic proteins including VGluT1, synaptophysin, Rab3A and synapsin with a lateral resolution of approximately 40 nm. Excitation multiplexing of suitable fluorescent dyes deciphered the spatial arrangement of the presynaptic phospho-protein synapsin relative to synaptic vesicles labeled with anti-VGluT1. Both predominantly occupied the same focal volume, yet may exist in exclusive domains containing either VGluT1 or synapsin immunoreactivity. While the latter have been observed with diffraction-limited fluorescence microscopy, STED microscopy for the first time revealed VGluT1-positive domains lacking synapsins. This observation supports the hypothesis that molecularly and structurally distinct synaptic vesicle pools operate in presynaptic nerve terminals.

Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy.

Human immunodeficiency virus type 1 (HIV-1) buds from the cell as an immature particle requiring subsequent proteolysis of the main structural polyprotein Gag for morphological maturation and infectivity. Visualization of the viral envelope (Env) glycoprotein distribution on the surface of individual HIV-1 particles with stimulated emission depletion (STED) superresolution fluorescence microscopy revealed maturation-induced clustering of Env proteins that depended on the Gag-interacting Env tail. Correlation of Env surface clustering with the viral entry efficiency revealed coupling between the viral interior and exterior: Rearrangements of the inner protein lattice facilitated the alteration of the virus surface in preparation for productive entry. We propose that Gag proteolysis-dependent clustering of the sparse Env trimers on the viral surface may be an essential aspect of HIV-1 maturation.

Parallelized STED fluorescence nanoscopy.

We introduce a parallelized STED microscope featuring m = 4 pairs of scanning excitation and STED beams, providing m-fold increased imaging speed of a given sample area, while maintaining basically all of the advantages of single beam scanning. Requiring only a single laser source and fiber input, the setup is inherently aligned both spatially and temporally. Given enough laser power, the design is readily scalable to higher degrees of parallelization m.

Solvation effects in the quartz crystal microbalance with dissipation monitoring response to biomolecular adsorption. A phenomenological approach.

Quartz crystal microbalance with dissipation monitoring (QCM-D) has become a popular tool to investigate biomolecular adsorption phenomena at surfaces. In contrast to optical mass-sensitive techniques, which commonly detect the adsorbed nonhydrated mass, the mechanically coupled mass measured by QCM-D includes a significant amount of water. A mechanistic and quantitative picture of how the surrounding liquid couples to the deposited solutes has so far been elusive for apparently simple phenomena like the random adsorption of nanometer-sized particles on a planar surface. Using a setup that enables simultaneous measurements by reflectometry and QCM-D on the same support, we have quantified the variations in coupled water, as sensed by the QCM frequency response, as a function of coverage for the formation of monolayers of globular proteins, virus particles, and small unilamellar vesicles. We found a close-to-linear relationship between the surface coverage and the relative contribution of water to the frequency response for these adsorption scenarios. The experimental hydration curves could be reproduced quantitatively using a theoretical model that assigns a pyramid-shaped hydration coat to each adsorbed particle and that accounts for the random distribution of adsorbents on the surface. This simple model fits the experimental data well and provides insight into the parameters that affect hydration.