Fig. 3.

M-STED of transcription foci within intact MCF10A nuclei. a Example of acquired stack images, from the confocal (F₁) to maximum STED power (F₈) (P_max = 43.3 mW). Color scale: normalized intensity. Scale bar: 3 µm. b Phasor plot obtained from the data and c the corresponding modulation image M(x,y). Color scale: modulation M (0.16–0.51). Scale bar: 500 nm. d F_in and F_out images obtained by application of SPLIT to F₈. Color scale: normalized intensity. Scale bar: 500 nm. e Comparison of the ability to separate two foci within intact nuclei between F₈ (black circles) and F_in (red diamonds), and the relation with the information encoded in M(x,y). f–j Quantification of the size of the transcription foci. f, g Example of data used for size estimation acquired at P_max = 24.2 mW. Shown are the sum of the full-size M-STED stack (f) and two ROIs selected for the analysis (g). Scale bars: 3 µm in f; 1 µm in g. h F_in images obtained from application of SPLIT to the ROIs, depicting resolved foci. Numbers indicate the estimated effective PSF size relative to the confocal (w/w_c). Scale bar: 1 µm. i Apparent size of individual foci shown in h. Scale bar: 1 µm. j Average Σ² values (Eq. (5)) extracted from each image such as represented in i (red) and from simulated images of point-like structures (black) shown in Supplementary Fig. 8. From the intercept (red), the size of the transcription foci was estimated to be 69 ± 5 nm