Plan apo objective lens

Cells grown on 18-mm square high-performance coverslip (No. 1.5, Zeiss) for 24 h were washed with PBS before fixing in 4% formaldehyde in PBS at 37 °C for 15 min. Subsequent permeabilisation, blocking, staining and mounting steps were as for regular immunofluorescence (see above). SIM was performed on a Zeiss Elyra PS1 microscope at 23 °C using a 63 × 1.4 N.A. plan-apo objective lens (Zeiss) and Immersol 518F (Zeiss) immersion oil. Image stacks were acquired using the Zeiss ZEN Black 2012 software for five grating phases and five grating rotations at z positions spaced 110 nm apart. Channel alignment information was created using a 3D array of multispectral beads imaged with the same instrument settings. Structured illumination processing and channel alignment were performed using the ZEN Black ELYRA edition software. The presented data are a region of a single slice out of a z-stack.

To determine cell viability after imaging experiments we used trypan blue^{44 (link)}. Specifically, we added 0.5 ml of 0.4% trypan blue (Sigma-Aldrich, St. Louis, MO) per well. The cell mono-layers were stained for 1 min. After staining, the cells were rinsed three times with PBS imaged and counted under the light microscope (PrimoVert, Carl Zeiss Microscopy, Peabody, MA) equipped with using 20X/0.3 NA PlANAPO objective lens (Zeiss, Oberkochen, Germany). Supplementary Fig. S5 presents example bright field images of dead and live cells after trypan blue staining. MB stained cells were light blue. The cells stained with both MB and trypan blue were purple and were considered dead.