Coherent-hybrid STED: a tunable photo-physical pinhole for super-resolution imaging at high contrast

Abstract

Resolution in microscopy is not limited by diffraction as long as a nonlinear sample response is exploited. In a paradigmatic example, stimulated-emission depletion (STED) fluorescence microscopy fundamentally 'breaks' the diffraction limit by using a structured optical pattern to saturate depletion on a previously excited sample area. Two-dimensional (2D) STED, the canonical low-noise STED mode, structures the STED beam by using a vortex phase mask, achieving a significant lateral resolution improvement over confocal fluorescence microscopy. However, axial resolution and optical sectioning remain bound to diffraction. Here we use a tunable coherent-hybrid (CH) beam to improve optical sectioning, markedly reducing background fluorescence. CH-STED, which inherits the 2D-STED immunity to spherical aberration, diversifies the depletion strategy, allowing an optimal balance between two key metrics (lateral resolution and background suppression) to be found. CH-STED is used to perform high-contrast imaging of complex biological structures, such as the mitotic spindle and the neuron cell body.

An anaphase surveillance mechanism prevents micronuclei formation from frequent chromosome segregation errors

“…These findings are at odds with the live-cell data presented here and are difficult to reconcile with the fact that Mklp2 is required for Kif4A accumulation at the spindle midzone (Nunes Bastos et al, 2013; see also Figures S3E-S3G), and Kif4A mediates Aurora B recruitment to this location (Kurasawa et al, 2004). Moreover, our close inspection of spindle midzone MTs in the absence of Mklp2 by super-resolution CH-STED microscopy (Pereira et al, 2019) failed to detect significant differences in MT density in the vicinity of anaphase lagging chromosomes (as well as any significant effect on non-KT MT half-life and spindle elongation capacity, as inferred from spinning disk confocal recordings), in agreement with recent expansion microscopy analysis of human cells depleted of PRC1, which is required for Kif4A recruitment to midzone MTs (Kurasawa et al, 2004;Vuku si c et al, 2021). One possibility is that Kif4A spatially controls the completion of NER on lagging chromosomes by (G) Frequency of mitotic errors that are positive for Nup153 in control and siMklp2-depleted cells (control n = 126 lagging, n = 5 bridges, n = 23 MN; siMklp2 n = 120 lagging, n = 4 bridges, n = 18 MN; data were pooled from at least 3 independent experiments, analyzed using the Fisher's exact two-tailed test; *p % 0.05, ***p % 0.001, and ****p % 0.0001).…”

“…We found that Nup153 recruitment to lagging chromosomes after Mklp2 RNAi could not be explained by the disassembly of midzone MTs, as midbodies with high MT density were clearly present and found to co-localize with MN that recruited Nup153 (Figures 7A and 7B; Video S7). Quantitative super-resolution Coherent-Hybrid Stimulated Emission Depletion (CH-STED) analysis in fixed cells (Pereira et al, 2019) confirmed no detectable differences in the density of midzone MT bundles surrounding anaphase lagging chromosomes, with or without Mklp2 (Figures 7C and 7D). We concluded that a midzonedependent Aurora B phosphorylation gradient, rather than midzone MT density, delays the completion of NER on lagging chromosomes to prevent MN formation in human cells.…”

Kinetochore-mediated microtubule assembly and Augmin-dependent amplification drive k-fiber maturation in mammals

“…The copyright holder for this preprint this version posted August 18, 2021. ; https://doi.org/10.1101/2021.08.18.456780 doi: bioRxiv preprint To gain additional insight into the role of Augmin in k-fiber formation, we optimized fixation conditions to preserve microtubule structure (see Materials and Methods) and inspected HAUS6-depleted cells by super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy, which markedly improves contrast in complex 3D objects like the mitotic spindle relative to conventional 2D-STED (Pereira et al, 2019). This analysis confirmed the absence of robust k-fibers after Augmin perturbation (Fig.…”

“…For Coherent-Hybrid STED (CH-STED) imaging, an Abberior 'Expert Line' gated-STED microscope was used, equipped with a Nikon Lambda Plan-Apo 1.4NA 60x objective lens. CH-STED was implemented as described before [42]. All acquisition channels (confocal and CH-STED) were performed using a 0. volumes that are assumed to contain well-defined microtubule populations were defined.…”

“…Here we used RNAi and high-resolution live-cell microscopy to investigate the role of 64 conserved mitotic proteins in mitotic spindle assembly and chromosome segregation in Indian muntjac cells. Assisted by unprecedented subsecond livecell super-resolution CH-STED nanoscopy analysis [42] of microtubule growth within individual k-fibers and direct perturbation of k-fiber structure by laser microsurgery, we identified Augmin as the main driver of k-fiber maturation. This function was distinct from the role of the Ndc80 complex in the stabilization of end-on kinetochore-microtubule attachments and independent of pioneer centrosomal microtubule scaffolds.…”