S plan fluor elwd 40 ph2 adm objective

Images were acquired with a Nikon A1R confocal laser scanning microscope (Nikon Instruments) mounted on a Nikon Ti-E inverted epifluorescence body with an S Plan Fluor ELWD 40 × Ph2 ADM objective (NA 0.60). Different fluorescent images were acquired along the z-axis to create a picture of the complete cell. EGFP was excited with a 488 nm argon ion laser and a 515–530 nm emission filter was used. Image analysis was conducted in Fiji (Schindelin et al., 2012 (link)) and the JaCoP tool (Bolte and Cordelières, 2006 (link)) was used for colocalization analysis.

Confocal images (16 bit) were acquired on a Zeiss LSM880 confocal microscope with Airyscan detector (Zeiss, Jena, Germany) or with a Nikon A1R confocal laser scanning microscope (Nikon Instruments) mounted on a Nikon Ti-E inverted epifluorescence body. A Plan-Apochromat 63×/1.4 oil objective was used to image in SR mode at a pixel size of 40 nm by 40 nm (Zeiss LSM880 confocal microscope) or an S plan Fluor ELWD 40× Ph2 ADM objective (NA 0.6) or CFI Plan Apo VC 60× WI DIC (NA 1.2) for the Nikon A1R confocal laser scanning microscope. EGFP was excited by the 488 nm laser line of an argon laser. A double band pass filter (BP 495-550) was placed in front of the Airyscan detector to detect EGFP emission (Zeiss LSM880 confocal microscope) or the emission filter was 515-530 nm for EGFP (Nikon A1R confocal laser scanning microscope). PI was excited with the 561 nm laser line of a diode laser. To detect PI emission, a combination of a BP 570-620 with a LP 645 filter was inserted in the light path. Images acquired with the Zeiss LSM880 confocal microscope were calculated through pixel reassignment and Wiener filtering by using the built-in ‘Airyscan Processing’ command in the Zen software. The images acquired with the Nikon A1R confocal laser scanning microscope were analyzed with Fiji software [59 (link)].