Switching behaviour of dSTORM dyes in glycerol-containing buffer.

To suppress optical aberrations caused by refractive index mismatch, we employ glycerol-immersion objectives in conjunction with fused silica cover glasses and imaging buffers with a high glycerol content. Here we demonstrate that the addition of glycerol to the buffer does not degrade the switching behaviour of the dyes Alexa Fluor 647 and Alexa Fluor 568 in dSTORM measurements, which shows that this approach is suitable for dSTORM. Additionally, we report evidence that sealed sample geometries as used in our experiments reduce photobleaching due to the lower influx of oxygen into the imaging buffer.

Intrinsic refractive index matched 3D dSTORM with two objectives: Comparison of detection techniques.

We have built a setup for 3D single molecule localisation microscopy (SMLM) where a very high resolution is achieved by, firstly, the use of two objectives instead of one and, secondly, minimizing optical aberrations by refractive index matching with a glycerol-water mixture as immersion medium in conjunction with glycerol-immersion objectives. Multiple optical paths of the microscope allow to switch between astigmatic and interferometric localisation along the optical axis, thus enabling a direct comparison of the performance of these localisation methods.

Bacterial twitching motility is coordinated by a two-dimensional tug-of-war with directional memory.

Type IV pili are ubiquitous bacterial motors that power surface motility. In peritrichously piliated species, it is unclear how multiple pili are coordinated to generate movement with directional persistence. Here we use a combined theoretical and experimental approach to test the hypothesis that multiple pili of Neisseria gonorrhoeae are coordinated through a tug-of-war. Based on force-dependent unbinding rates and pilus retraction speeds measured at the level of single pili, we build a tug-of-war model. Whereas the one-dimensional model robustly predicts persistent movement, the two-dimensional model requires a mechanism of directional memory provided by re-elongation of fully retracted pili and pilus bundling. Experimentally, we confirm memory in the form of bursts of pilus retractions. Bursts are seen even with bundling suppressed, indicating re-elongation from stable core complexes as the key mechanism of directional memory. Directional memory increases the surface range explored by motile bacteria and likely facilitates surface colonization.