Live 2D-SIM (light modulation/grazing incidence illumination microscopy, GI-SIM) images of cells stained for 20 minutes with ER Tracker Green (ThermoFisher scientific, E34251) were acquired with a custom-built highspeed SIM microscope19 ,20 (link), using a spatial light modulator (SLM). ER Tracker Green was imaged using a 100X/1.49NA TIRF oil-immersion objective (Olympus) with a 488 nm diode laser (Toptica) at an irradiance of 50W/cm2, with emission imaged via a notch filter (FF01-525/30, Semrock) onto an sCMOS camera (ORCA Flash 4.0, Hamamatsu). 2D-SIM gratings displayed on the SLM resulted in a line spacing of 228 nm at the sample, corresponding to an angle of incidence of 44.6°. Each super-resolved frame was obtained from the reconstruction24 (link),28 (link) of nine raw frames acquired at 6 ms/exposure (54 ms/SIM frame). 3D-SIM (23 slices, 2.4 µm) was performed using Elyra microscope (Zeiss), with Plan Apochromat X63/1.4NA oil objective and sCMOS PCO Edge camera (Andor) on Paraforamdehyde 2%, Gluteraldehyde 2%, 100mM sodium cacodylate, 2mM CaCl2, pH7.4 1 hour room temperature. See Supplementary Note 1 for further details of SIM image analysis.
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High-speed 2D & 3D SIM Imaging of ER
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Corresponding Organization : UK Dementia Research Institute
Other organizations : Institut de Biologie de l'École Normale Supérieure, École Normale Supérieure - PSL
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independent variables
- Spatial light modulator (SLM) with 2D-SIM gratings to achieve a line spacing of 228 nm at the sample
- Angle of incidence of 44.6°
- Exposure time of 6 ms per raw frame (54 ms per SIM frame)
- 3D-SIM using a Plan Apochromat X63/1.4NA oil objective
- ER Tracker Green fluorescence signal
- ER Tracker Green staining for 20 minutes
- Irradiance of 50 W/cm^2 from a 488 nm diode laser
- Emission imaged via a notch filter (FF01-525/30, Semrock)
- SCMOS camera (ORCA Flash 4.0, Hamamatsu) used for image acquisition
- Paraformaldehyde 2%, Gluteraldehyde 2%, 100 mM sodium cacodylate, 2 mM CaCl2, pH 7.4 fixation for 3D-SIM
- Positive control: Not specified
- Negative control: Not specified
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