Here, we measure the actin cytoskeleton arrangement of different morphological states of human platelets using a new protocol for photo-switching of rhodamine class fluorophores. A new medium composition was established for imaging the cytoskeleton using Alexa Fluor 488 conjugated to phalloidin. Morphological states of platelets bound to a glass substrate are visualized and quantified by two-dimensional localization microscopy at nanoscopic resolution. Marker-less drift correction yields localization of individual Alexa 488 conjugated to phalloidin with a positional accuracy of 12 nm.

Over-expression of fluorescently-labeled markers for extracellular vesicles is frequently used to visualize vesicle up-take and transport. EVs that are labeled by over-expression show considerable heterogeneity regarding the number of fluorophores on single particles, which could potentially bias tracking and up-take studies in favor of more strongly-labeled particles. To avoid the potential artefacts that are caused by over-expression, we developed a genome editing approach for the fluorescent labeling of the extracellular vesicle marker CD63 with green fluorescent protein using the CRISPR/Cas9 technology. Using single-molecule sensitive fluorescence microscopy, we quantitatively compared the degree of labeling of secreted small extracellular vesicles from conventional over-expression and the CRISPR/Cas9 approach with true single-particle measurements. With our analysis, we can demonstrate a larger fraction of single-GFP-labeled EVs in the EVs that were isolated from CRISPR/Cas9-modified cells (83%) compared to EVs that were isolated from GFP-CD63 over-expressing cells (36%). Despite only single-GFP-labeling, CRISPR-EVs can be detected and discriminated from auto-fluorescence after their up-take into cells. To demonstrate the flexibility of the CRISPR/Cas9 genome editing method, we fluorescently labeled EVs using the HaloTag^® with lipid membrane permeable dye, JaneliaFluor^® 646, which allowed us to perform 3D-localization microscopy of single EVs taken up by the cultured cells.

Herein, we present a new algorithm for real-time analysis of 3D single molecule localization microscopy images with a small impact on fitting accuracy using lookup-tables with discrete xyz-positions. The algorithm realizes real-time visualization during acquisition. We demonstrate its performance on simulated and measured data. Additionally, combining real-time fitting with a feedback loop controlling the activation laser pulse keeps the number of emitters per image frame constant.

Human immune protection against bacteria critically depends on activation of the complement system. The direct bacteriolytic activity of complement molecules against Gram-negative bacteria acts via the formation of Membrane Attack Complex (MAC) pores. Bactericidal MAC pores damage the bacterial outer membrane, leading to destabilization of the inner membrane. Although it is well-established that inner membrane damage is crucial for bacterial cell death, the critical event causing MAC-mediated inner membrane damage remains elusive. Here we question whether the bacterial cell envelope possesses vulnerable spots for MAC pores to insert. By following the localization of MAC pores on E. coli over time using fluorescence microscopy, we elucidate that MAC deposition initiates at the new bacterial pole, which induces inner membrane damage and halts bacterial division. MAC components C8 and C9 preferentially localize at new bacterial poles, while C3b localizes randomly on the bacterial surface. This suggests that preferential MAC localization is determined by one of the initial steps of MAC formation. These findings provide valuable information about the interplay between immune components and the Gram-negative cell envelope.

In baker’s yeast (Saccharomyces cerevisiae), Trk1, a member of the superfamily of K-transporters (SKT), is the main K⁺ uptake system under conditions when its concentration in the environment is low. Structurally, Trk1 is made up of four domains, each similar and homologous to a K-channel α subunit. Because most K-channels are proteins containing four channel-building α subunits, Trk1 could be functional as a monomer. However, related SKT proteins TrkH and KtrB were crystallised as dimers, and for Trk1, a tetrameric arrangement has been proposed based on molecular modelling. Here, based on Bimolecular Fluorescence Complementation experiments and single-molecule fluorescence microscopy combined with molecular modelling; we provide evidence that Trk1 can exist in the yeast plasma membrane as a monomer as well as a dimer. The association of monomers to dimers is regulated by the K⁺ concentration.