1.518 refractive index oil

SIM was performed on a Zeiss LSM 780 Elyra PS1 microscope (Carl Zeiss, Germany) using C Plan-Apochromat 63×/1.4 oil objective with a 1.518 refractive index oil (Carl Zeiss). The fluorescence signal is detected on an EMCCD Andor Ixon 887 1 K. Raw images are composed of fifteen images per plane per channel (five phases, three angles), and acquired with a Z-distance of 0.11 μm. Acquisition parameters were adapted from one image to one other to optimize the signal to noise ratio. SIM images were processed separately for each channel with ZEN software and then corrected for chromatic aberration using 100-nm TetraSpeck microspheres (ThermoFisher Scientific) embedded in the same mounting media as the sample. The SIMcheck plugin in imageJ was used to analyze the quality of the acquisition and the processing in order to optimize parameters for resolution, signal-to-noise ratio, and reconstruction pattern^{63 (link)}.

The super-resolved images were acquired on a Elyra 7 Lattice SIM microscope (Carl Zeiss, Germany) using a 63×/1.46 oil alpha Plan Apo objective with a 1.518 refractive index oil (Carl Zeiss) and an sCMOS PCO Edge 4.2 camera for the detection. Thirteen images per plane per channel were acquired with a Z-distance of 0.101 μm to perform 3D-SIM images. The ZEN software was used to process the SIM images.

IMR-90 fibroblasts at PN16 and PN35 were cultured on precision glass coverslips thickness No. 1.5 H (Marienfeld Superior). Immunofluorescence was performed as indicated in the previous section, using Fluoromont-G (Thermo fisher Scientific) as mounting medium. SIM was performed on a Zeiss LSM 780 Elyra PS1 microscope (Carl Zeiss, Germany) using 63×/1.4 oil Plan Apo objective with a 1.518 refractive index oil (Carl Zeiss) and an EMCCD Andor Ixon 887 1 K camera for the detection. Fifteen images per plane per channel (five phases, three angles) were acquired with a Z-distance of 0.09 μm to perform 3D-SIM images. Acquisition parameters were adapted from one image to one other to optimize the signal to noise ratio. SIM images were processed with ZEN software and then aligned with ZEN using 100-nm TetraSpeck microspheres (ThermoFisher Scientific) embedded in the same conditions as the sample. The SIMcheck plugin in imageJ was used to analyze the acquisition and the processing in order to optimize for resolution, signal to noise ratio, and reconstruction pattern^{65 (link)}.