Assessing crosstalk in simultaneous multicolor single-molecule localization microscopy.

Single-molecule localization microscopy (SMLM) can reach sub-50 nm resolution using techniques such as stochastic optical reconstruction microscopy (STORM) or DNA-point accumulation for imaging in nanoscale topography (PAINT). Here we implement two approaches for faster multicolor SMLM by splitting the emitted fluorescence toward two cameras: simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) that images spectrally separated red and far-red imager strands on each camera, and spectral demixing dSTORM (SD-dSTORM) where spectrally close far-red fluorophores appear on both cameras before being identified by demixing. Using S2C-DNA-PAINT as a reference for low crosstalk, we evaluate SD-dSTORM crosstalk using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets.

Presynapses contain distinct actin nanostructures.

The architecture of the actin cytoskeleton that concentrates at presynapses remains poorly known, hindering our understanding of its roles in synaptic physiology. In this work, we measure and visualize presynaptic actin by diffraction-limited and super-resolution microscopy, thanks to a validated model of bead-induced presynapses in cultured neurons. We identify a major population of actin-enriched presynapses that concentrates more presynaptic components and shows higher synaptic vesicle cycling than their non-enriched counterparts.

Automating multimodal microscopy with NanoJ-Fluidics.

Combining and multiplexing microscopy approaches is crucial to understand cellular events, but requires elaborate workflows. Here, we present a robust, open-source approach for treating, labelling and imaging live or fixed cells in automated sequences. NanoJ-Fluidics is based on low-cost Lego hardware controlled by ImageJ-based software, making high-content, multimodal imaging easy to implement on any microscope with high reproducibility.