Plan apochromat 63 na 1.40 oil dic 2 objective

Confocal micrographs were obtained using an LSM710 or LSM780 confocal microscope (Carl Zeiss) equipped with an Ar-laser multiline (458/488/514 nm), a DPSS-561 10 (561 nm), a continuous-wave laser diode 405–30 CW (405 nm), and an HeNe laser (633 nm). The objective used was a Plan-Apochromat 63×/1.40 oil differential interference contrast (DIC) III (Carl Zeiss). Images were analyzed and adjusted (brightness/contrast) in ImageJ/Fiji (Schindelin et al., 2012 (link)) or Zen Blue. For superresolution microscopy, a Zeiss LSM 880 Airyscan (Carl Zeiss) was used with a Zeiss plan-apochromat 63× NA/1.40 oil DIC II objective (Carl Zeiss). The Airyscan detector was either in confocal or super-resolution mode, giving images with voxel size 0.0426 × 0.0426 × 0.1850 µm. Airyscan raw images were processed using Zen Blue and aligned in Zen Black. Images were further processed in ImageJ/Fiji (brightness/contrast; Schindelin et al., 2012 (link)) or 3D rendered using Imaris 7.7.2 (Bitplane). All images within one dataset were taken at fixed intensities below saturation, and identical settings were applied for all treatments within one experiment. In general, at least five (but often more) images were taken randomly throughout the coverslips. Manders colocalization coefficient was determined with the ImageJ plugin “JACoP” (Bolte and Cordelières, 2006 (link)). Specific analyses are described below.