Apo tirf 1.49 na oil objective

Cell separation phenotypes were assessed essentially as described (33 (link)), except that cells were stained with 8 μg/ml propidium iodide and 10 nM calcofluor white, both from Sigma. Prior to staining, cells were grown in EMM supplemented with 225 mg/L each of adenine, histidine, leucine, lysine, and uracil and grown to an OD600 of 0.4–0.5. Imaging was performed on a Nikon Eclipse Ti with a CSU-W1 Yokogawa disc. A 405/488/561/640 dichroic was used. 405 nm laser light was used to excite calcofluor white, and 561 nm laser light was used to excite propidium iodide. Emission for calcofluor white and propidium iodide was collected through 435–485 nm and 590–650 nm BP filters, respectively, using a Hamamatsu ORCA Flash 4.0 sCMOS camera. A Nikon 60× ApoTIRF 1.49 NA oil objective was used. Z-stacks were acquired with a spacing of 0.7 microns, and a max projection was applied prior to image analysis. At least 500 cells per genotype were scored manually for the number of septa per cell. Samples were blinded before imaging and only unblinded after determining the number of cells with no, one or two or more septa.