Fig. 1: Correlative quantification of diffusing assemblies using SlimVar.

Terms are defined in Table 1. SlimVar delivers a rapid photobleaching in image sequences at high (millisecond) framerate, over ~10 s cumulative exposure time t, to outpace molecular diffusion; followed by b robust postprocessing and quality control steps to identify foci in individual frames, and tracks across multiple (up to 20) frames, which correspond in general to assemblies of labelled molecules. c The full extent of photobleaching enables estimates of the characteristic molecular brightness (red arrows), which is narrowly distributed for a fluorescent protein. The characteristic molecular brightness is used to determine d total protein number for each region of interest and e stoichiometry for each tracked assembly near the start of the image sequence (blue arrows), as a number of molecules (red circles). These metrics are corrected for autofluorescence using unlabelled wild type then collated over a population, enabling robust estimation for average total protein number. f Periodicity analysis extracts patterns from the stoichiometry distribution to infer consistent repeat units of assemblies (dark circles). g Rapid tracking facilitates analysis of mean-square displacements to estimate individual assembly mobility. h Multicolour SlimVar assesses whether stoichiometry and diffusivity are dependent on colocalisation between different pairs of assemblies (white overlap between individual channels in green and magenta).