MINFLUX reveals dynein stepping in live neurons.

Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons.

Neurofilament Levels in Dendritic Spines Associate with Synaptic Status.

Neurofilaments are one of the main cytoskeletal components in neurons; they can be found in the form of oligomers at pre- and postsynapses. How their presence is regulated at the postsynapse remains largely unclear. Here we systematically quantified, by immunolabeling, the occurrence of the neurofilament isoform triplet neurofilament light (NFL), medium (NFM), and heavy (NFH) at the postsynapse using STED nanoscopy together with markers of synaptic strength and activity.

Robust nanoscopy of a synaptic protein in living mice by organic-fluorophore labeling.

Extending superresolution fluorescence microscopy to living animals has remained a challenging frontier ever since the first demonstration of STED (stimulated emission depletion) nanoscopy in the mouse visual cortex. The use of fluorescent proteins (FPs) in in vivo STED analyses has been limiting available fluorescence photon budgets and attainable image contrasts, in particular for far-red FPs. This has so far precluded the definition of subtle details in protein arrangements at sufficient signal-to-noise ratio.