SignificanceLassa virus is a highly pathogenic arenavirus that causes severe hemorrhagic fever in humans. Currently, there are no efficacious vaccines or treatments available to combat this pathogen. An important component of any vaccine candidate against Lassa virus will likely include the highly glycosylated glycoprotein complex presented on the virion surface. Here, we determine the composition of the Lassa virus glycome, revealing that the virus presents an abundance of glycans that are not biosynthetically processed to full maturity. Such underprocessed glycans form spatially distinct clusters, which shield the proteinous surface of the Lassa virus glycoprotein spike from the humoral immune response. These data are integral for the development of humoral-based vaccines that mimic the mature Lassa virion.

Correlative light and electron cryo-microscopy (cryo-CLEM) combines information from the specific labeling of fluorescence cryo-microscopy (cryo-FM) with the high resolution in environmental context of electron cryo-microscopy (cryo-EM). Exploiting super-resolution methods for cryo-FM is advantageous, as it enables the identification of rare events within the environmental background of cryo-EM at a sensitivity and resolution beyond that of conventional methods. However, due to the need for relatively high laser intensities, current super-resolution cryo-CLEM methods require cryo-protectants or support films which can severely reduce image quality in cryo-EM and are not compatible with many samples, such as mammalian cells. Here, we introduce cryogenic super-resolution optical fluctuation imaging (cryo-SOFI), a low-dose super-resolution imaging scheme based on the SOFI principle. As cryo-SOFI does not require special sample preparation, it is fully compatible with conventional cryo-EM specimens, and importantly, it does not affect the quality of cryo-EM imaging. By applying cryo-SOFI to a variety of biological application examples, we demonstrate resolutions up to ∼135 nm, an improvement of up to three times compared with conventional cryo-FM, while maintaining the specimen in a vitrified state for subsequent cryo-EM. Cryo-SOFI presents a general solution to the problem of specimen devitrification in super-resolution cryo-CLEM. It does not require a complex optical setup and can easily be implemented in any existing cryo-FM system.

Gold nanoparticles (GNPs) enhance the damaging absorbance effects of high-energy photons in radiation therapy by increasing the emission of Auger-photoelectrons in the nm-μm range. It has been shown that the incorporation of GNPs has a significant effect on radiosensitivity of cells and their dose-dependent clonogenic survival. One major characteristic of GNPs is also their diameter-dependent cellular uptake and retention. In this article, we show by means of an established embodiment of localization microscopy, spectral position determination microscopy (SPDM), that imaging with nanometer resolution and systematic counting of GNPs becomes feasible, because optical absorption and plasmon resonance effects result in optical blinking of GNPs at a size-dependent wavelength. To quantify cellular uptake and retention or release, SPDM with GNPs that have diameters of 10 and 25 nm was performed after 2 h and after 18 h. The uptake of the GNPs in HeLa cells was either achieved via incubation or transfection via DNA labeling. On average, the uptake by incubation after 2 h was approximately double for 10 nm GNPs as compared to 25 nm GNPs. In contrast, the uptake of 25 nm GNPs by transfection was approximately four times higher after 2 h. The spectral characteristics of the fluorescence of the GNPs seem to be environment-dependent. In contrast to fluorescent dyes that show blinking characteristics due to reversible photobleaching, the blinking of GNPs seems to be stable for long periods of time, and this facilitates their use as an appropriate dye analog for SPDM imaging.