Na super plan apochromat oil objective

A DeltaVision RT system (Olympus IX71 based; Applied Precision Ltd.) equipped with a Photometrics CoolSnap HQ camera (Roper Scientific), a 100×/1.4-NA Super-Plan Apochromat oil objective (Olympus), a four-color Standard Insight SSI module light source, a workstation with a CentOS operating system, and softWorx software (Applied Precision Ltd.) was used for imaging at room temperature (23°C). Cells were placed on a microscopic slide and kept in their growth media (live cells) or PBS (fixed cells) for the time of imaging. For all fluorescence signal quantification experiments, all imaging was conducted with the same exposure and illumination settings on living cells to allow the direct comparison of the results. For the quantification of the signal intensity, the integrated density (IntDen) of the SPB (Spc42-yeGFP) in the brightest stack was measured with a 5 × 5 pixel-square and a 7 × 7 pixel-square for background correction. The following formula was used to calculate the relative fluorescence intensity: RFI = IntDen_5×5 – {[IntDen_7×7 – IntDen_5×5] × [area_5×5/(area_7×7 – area_5×5)]}. Image processing and phenotypic analysis were performed with ImageJ software. Quantifications were performed three times and analyzed with GraphPad Prism software. A combined graph is shown in Fig. 4, E and F; Fig. 5, C and D; and Fig. 6 C.

A DeltaVision RT system (Olympus IX71 based; Applied Precision Ltd.) equipped with a Photometrics CoolSnap HQ camera (Roper Scientific), a 100×/1.4-NA Super-Plan Apochromat oil objective (Olympus), a four-color Standard Insight SSI module light source, a workstation with a CentOS operating system, and softWoRx software (Applied Precision Ltd.) was used for cell imaging. Imaging was done at 30°C or 37°C with GFP or mCherry channels. The imaging was conducted with the same exposure and illumination setting to allow direct comparison of the results, and one single stack was used for the analysis. Images were deconvolved with softWoRx software (Applied Precision) and processed with ImageJ (National Institutes of Health, Bethesda, MD). Imaging experiments and quantifications were performed three times and analyzed with GraphPad Prism software.