Airyscan super resolution gaasp detector

Confocal fluorescence microscopy was performed as previously described [77 (link)]. Super-resolution fluorescence microscopy was performed on a Zeiss LSM 880 Confocal microscope with an Airyscan super-resolution GaAsP detector and 63x/1.4 oil immersion objective (Zeiss, Oberkochen, Germany). All fluorophores were acquired using pixel dwell times at 1.31 μs per pixel. GFP-Opi1p, Tcb3p-GFP and DsRed-HDEL were excited using a 488 nm and 561 nm lasers, respectively. Relative laser intensities for 488 nm laser and 561 nm laser were both set to 1.5 and the digital gain was set to 900. Images were Airyscan processed in Zen Black and deconvolved in Zen Blue using fast-iterative deconvolution (Zeiss Canada, North York, ON, Canada). Cortical association was assayed by tracing the cell cortex in the Zen Blue profile mode, then measuring cortical fluorescence intensity at the cortex. Cortical association was expressed as a ratio of the total distance of cortical fluorescence to the total cortical distance. Images were exported as 8-bit uncompressed TIFF files then processed in Affinity Photo (Serif Ltd., Nottingham, UK).

Superresolution fluorescence microscopy was performed on a Zeiss LSM 880 confocal laser scanning microscope with an Airyscan superresolution GaAsP detector and 63×/1.4 oil immersion objective (Zeiss). All fluorophores were acquired using pixel dwell times at approximately 1.31 μsec per pixel. DsRed-HDEL fluorescence was excited using a 561-nm laser, and GFP fusions were excited using a 488-nm laser. Relative intensities were set to 1.5 for both lasers. Digital gain was set to 900 for the 488-nm laser and 800 for the 561-nm laser. Images were Airyscan processed in Zen Black and deconvolved in Zen Blue (Zeiss). ER association with the PM was determined by tracing the cell cortex in the Zen Blue profile mode, then measuring cER fluorescence intensity at the cortex. ER association with the cell cortex was expressed as a ratio of the total distance of cER fluorescence to the total PM perimeter. Images were exported as 8-bit uncompressed TIFF files then processed in Affinity Photo (Serif Ltd). Contrast enhancement was kept constant for each series of images. Levels of PM Lact-C2-GFP fluorescence were quantified using ImageJ (https://imagej.nih.gov/ij/index.html) by determining the mean fluorescence of selected areas corresponding to the cell cortex.