Fig 2: Two-colour super-resolution imaging of nucleolar sub-domains. (A) HeLa cells granular component (GC) was imaged with the help of NPM protein fused to mEos2 (green). The fibrillar centres (FCs) were imaged by immunostaining RPA proteins with A647-Ab (magenta). (B) HeLa cells GC was visualized together with the dense fibrillar component (DFC), by using mEos2-NPM in combination with Fib proteins immunostained by A647-Ab (magenta). (C) Nucleolar organization of human HeLa cell observed by electron microscopy. Reprinted with permission of the author and the editor (permission is granted to Macmillan Publishers Ltd, part of Springer Nature)31. (D) Ring-like structure of DFC revealed by super-resolution localization microscopy (zoom of the white ROI in B). The inner diameter (FWHM = 160 nm) and the thickness (FWHM = 75 nm) of the ring were measured from the cross section displayed in inset. All super-resolution images were obtained from a 20000 images stack as described in the main text. Scale bars: 1 µm.

Fig 3: Confocal microscopy imaging of nucleolar sub-domains in HeLa cells. Column 1: NPM protein fused to the fluorescent protein eGFP was overexpressed after cell transient transfection in order to image the nucleoli granular component (A1). Endogenous RPA was immunolabelled with A647-Ab (B1). The merged images evidenced the specificity of the labelling strategy (C1). Column 2: eGFP-NPM localization is displayed in panel A2. Endogenous Fib was immunolabelled with A647-Ab (B2). The merged images evidenced the spatial overlap between the two domains (C2). Column 3: Endogenous Fib was immunolabelled with A555-Ab (A3). Endogenous RPA was immunolabelled with A647-Ab (B3). The merged images evidenced the spatial overlap between the two domains (C3). Scale bar: 5 µm.