Figure 3

3D-SIM imaging of mouse spleen tissue sections. 10 µm-thick tissue sections were stained for nuclear DNA using Hoechst 33258. Subsequently, they were embedded in different mounting media in order to equalise its optical properties, decrease light scattering, and improve quality of 3D-SIM images. 12 µm-thick orthogonal cross-sections (XZ) through 3D-SIM images demonstrate axial and horizontal resolution achieved in specimen embedded in Vectashield (A), S65VE (B), F80VE (C), Sorb70VE (D) and H71VE (E). 0 µm corresponds to an imaging plane closest to the coverslip. For the sake of clarity the values of respective refractive indices and MCNR are given. Enlarged insets are provided on the right-hand side. Scale bar equals to 3 µm. White arrowheads in B–E indicate interruptions in the nuclear periphery unravelled due to enhanced resolution. Intensity histogram was adjusted to min-max. (F) Bar-plot presenting median values of MCNR scored for various clearing media (at least 3 measurements). Error bars correspond to the standard deviation. (G) Graph demonstrates radial profiles of averaged frequency spectra obtained for several raw 3D-SIM images. Same colour coding was used as in F. Original frequency spectra provided in Supplementary Fig. S6A. Inset presents an example of averaged frequency spectrum obtained for mouse spleen sections embedded in H71VE. Yellow arrow demonstrates the direction of intensity integration. Black arrowheads point at peaks corresponding to SIM illumination pattern frequencies.