Structured Illumination Microscopy Protocol

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)}.

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Addi C., Presle A., Frémont S., Cuvelier F., Rocancourt M., Milin F., Schmutz S., Chamot-Rooke J., Douché T., Duchateau M., Giai Gianetto Q., Salles A., Ménager H., Matondo M., Zimmermann P., Gupta-Rossi N, & Echard A. (2020). The Flemmingsome reveals an ESCRT-to-membrane coupling via ALIX/syntenin/syndecan-4 required for completion of cytokinesis. Nature Communications, 11, 1941.

Publication 2020

Zeiss LSM 780 Elyra PS1 microscope 1.518 refractive index oil TetraSpeck microspheres

Fluorescence Microscope Microspheres Reconstruction

Corresponding Organization :

Other organizations : Institut Pasteur, Centre National de la Recherche Scientifique, Inserm, Centre de Recherche en Cancérologie de Marseille, Aix-Marseille Université

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Variable analysis

independent variables

Acquisition parameters were adapted from one image to one other to optimize the signal to noise ratio.

dependent variables

Resolution
Signal-to-noise ratio
Reconstruction pattern

control variables

Zeiss LSM 780 Elyra PS1 microscope
C Plan-Apochromat 63×/1.4 oil objective
1.518 refractive index oil
EMCCD Andor Ixon 887 1 K detector
Z-distance of 0.11 μm
100-nm TetraSpeck microspheres embedded in the same mounting media as the sample

controls

100-nm TetraSpeck microspheres embedded in the same mounting media as the sample

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