Cfi plan apo lambda 100x

Schizosaccharomyces pombe (S. pombe) cells were grown in YES (yeast extract with supplements) medium overnight at 30°C. Prior to imaging, 1 ml S. pombe cell culture with OD_595nm 0.4-0.6 was concentrated to 50 µl after centrifugation at 1500 g for 30 s. 2 µl of cell suspension were loaded under a 22×22 mm glass coverslip (VWR, thickness: 1.5). Spinning disk confocal images of S. pombe were captured with an Eclipse Ti-E inverted microscope fitted with a Yokogawa CSU-X1 spinning disk confocal scanning unit, 600 series SS 488 nm, SS 561 nm lasers, single band filters FF01-525/50-25 and FF01-617/73-25 (Semrock Brightline), Nikon CFI Plan Apo Lambda 100x (NA=1.45) oil objective and an Andor iXon Ultra U3-888-BV monochrome EMCCD camera. Image acquisition was controlled by Andor IQ3 software.

Images of GUVs labeled with Fast DiO and live cells were obtained using Yokogawa CSU-X1 spinning disk confocal system mounted on the Eclipse Ti-E Inverted microscope with Nikon CFI Plan Apo Lambda 100X Oil N.A. = 1.45 oil objective, 600 series SS 488nm, SS 561nm lasers and Andor iXon Ultra U3-888-BV monochrome EMCCD camera controlled by Andor IQ3. Maximum intensity projections of 8–10 z stacks of 0.5 μm step size images are shown in Figures 4D, 4E, and S5G. For Figures 5C, 5D, S5D, and S5E single plane images are presented. Fluorescence images are shown with inverted LUT (look-up table).
Image processing and quantifications were performed in Fiji [67 (link)]. Images within each experiment images are shown within the same display range. Calibration bars are included. For Figure 4D average fluorescence intensity was measured per cell and normalized to the control. To measure the number of Golgi cisternae (Figure 4E), maximum intensity projections of spinning disk confocal images were thresholded. Cisternae numbers were then normalized to cell area.