Fig. 3: SlimVar resolves dynamics of VIN3 and VRN5 assemblies during cold exposure of root tips.

a Schematic of whole roots laid horizontally in media between agarose and coverslip for confocal and SlimVar microscopy. Created in BioRender. Payne-Dwyer, A. (2025) https://BioRender.com/13pyw8b. b Projected confocal z-stacks of VRN5-YFP root tips after 6 weeks of cold; acquisition time 35 s. Insets (interpolated) show VRN5 consistently localised to the nucleoplasm but not the nucleolus. Patterning of VEL proteins appeared round or lens-sh. aped (c.f. Supplementary Fig. 9), with median length 7.8 μm (interquartile range IQR: 5.7–10.3 μm, N = 571), and aspect ratio 1.16 (IQR: 1.06–2.10), comparable to nuclear reporters⁷⁸. c, d Airyscan images of VRN5-YFP after 2 weeks’ cold indicating heterogeneous distribution, shared scale bar 2 µm; c maximum intensity projection of three z-slices, averaged over three consecutive timepoints; d residence times estimated from the ratio between median and standard deviation of pixelwise values across three frames. Low standard deviation (cyan) indicates low displacement of foci over 200 ms, equivalent to diffusivity <0.1 µm²/s, while high standard deviation (magenta) indicates high displacement over 70 ms, or diffusivity >0.3 µm²/s. e Schematic indicating illumination and detection volumes (highlighted region and red box, respectively) and working depth. f–k SlimVar images of a VRN5-YFP root tip before vernalisation; shared scale bar 5 µm. f Brightfield for identifying and centring nuclei; g initial fluorescence frame, with nucleolus indicated (white dashes) and overlapping signals; h photobleaching transiently increases contrast, revealing distinct assemblies (mean projection of frames 4–6); i SlimVar resolves assemblies of different mobility on ms timescales, shown as distinct slow- (cyan, >60 ms residence time, diffusivity <0.4 µm²/s) and fast-moving (magenta, <20 ms residence time, diffusivity >1.4 µm²/s) objects, represented by pixelwise ratio of median and standard deviation. j Foci are detected from local maxima to super-resolved localisation precision. All sifted foci (Methods) for full sequence shown superimposed (white circles) on panel h (greyscale); k tracks, generated by linking nearby foci, indicate individual assemblies with independent estimates of stoichiometry and diffusivity. All sifted tracks from sequence shown with one vertex per timepoint (white arrows).