Resolution in optical nanoscopy depends on the localization uncertainty of single fluorescent labels, the density of labels covering the sample, and the sample’s spatial structure. Currently there is no integral, practical resolution measure that takes all factors into account. Here we introduce such a measure that can be computed directly from the image. We demonstrate its validity and benefits on 2D and 3D localization microscopy images of tubulin and actin filaments. Our approach makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, optimize and rank different emitter localization and labeling strategies, define a stopping criterion for data acquisition, describe image anisotropy and heterogeneity, and, surprisingly, estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, but instead show how the best image resolution can be achieved as fast as possible.

A lack of techniques to image multiple genomic loci in living cells has limited our ability to investigate chromosome dynamics. Here we describe CRISPRainbow, a system for labeling DNA in living cells based on nuclease-dead (d) Cas9 combined with engineered single guide RNA (sgRNA) scaffolds that bind sets of fluorescent proteins. We demonstrate simultaneous imaging of up to six chromosomal loci in individual live cells and document large differences in the dynamic properties of different chromosomal loci.

Export of mRNA occurs via nuclear pores, large nano-machines with diameters of roughly 120 nm that are the only link between nucleus and cytoplasm1. Hence, mRNA export occurs over distances smaller than the optical resolution of conventional light microscopes. There is extensive knowledge on the physical structure and composition of the NPC2–7, but transport selectivity and dynamics of mRNA export at nuclear pores remain unknown8. We developed a super-registration approach using fluorescence microscopy that can overcome the current limitations of colocalization by means of measuring intermolecular distances of chromatically different fluorescent molecules with nm precision. With this method we achieve 20 ms time- and at least26 nm spatial precision, rendering the capture of highly transient interactions in living cells possible. With this method we were able to spatially resolve the kinetics of mRNA transport and present a three step model consisting of docking (80ms), transport (5–20ms) and release (80ms), totalling 180 ± 10 ms. Importantly, the translocation through the channel was not the rate-limiting step, mRNAs can move bi-directionally in the pore complex and not all pores are equally active.

How CRISPR Cas9–guide RNA complexes navigate the nucleus and interrogate the genome is not well understood. Ma et al. track these complexes in live cells and find that mutations in the guide seed region significantly reduced the complex’s target residence time, with a commensurate impairment of cleavage.

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