Ix 71 microscope

Drosophila cell lines were grown in Express-Five medium (GIBCO). The AC5-Polo-GFP cell line was described previously [26 (link)]. Cells exponentially growing were seeded on Con-A treated coverslips and treated with either DMSO or 100 nM BI 2536 for 2 h before being processed for immunostaining as described previously [23 (link),25 (link)]. Imaging was performed using an Olympus IX-71 microscope controlled by Delta Vision SoftWorx (Applied Precision, Issequa, WA, USA). Image stacks were deconvolved, quick-projected, and saved as tiff images to be processed using Adobe PhotoShop.
Signal intensities were measured using the softWoRx Data Inspector tool; average background was subtracted; data were plotted using Prism software.

All images were acquired using an inverted Olympus IX-71 microscope controlled by the Deltavision Elite Imaging System (Applied Precision) equipped with a Photometric CoolSNAP HQ² CCD camera. Images from individual metaphase spreads were collected at the same exposure time using a 40× objective. Deconvolved images (conservative ratio, 10 iterations) were projected and saved as Photoshop and TIF files. Fiber images were collected using the 100× objective, and those fibers extending through multiple fields of view were captured using the Panels option in the softWoRx Acquire 3D program and merged into single images using the ‘Stitch’ function. All images were exported for analysis into Adobe Photoshop and ImageJ.

To analyze the filamentation capability of U. maydis in PD-charcoal plates, single colonies where visualized using the Leica M205 Stereoscope equipped with an ORCA-Flash4.0 LT Hamamatsu digital camera. The area of the colonies was measured by selecting the perimeter of each colony using the plugging convex hull of ImageJ software. To determine sirtuins’ localization, sir2:eGFP, hst2:eGFP, hst4:eGFP, hst5:eGFP and hst6:eGFP cells were visualized using a DeltaVision microscopy system comprising an Olympus IX71 microscope and CoolSnap HQ camera (Applied Precision, Issaquah WA, United States). To visualize mitochondria, 0.5 mM Mito-Tracker CM-H2Xros (Molecular Probes, Eugene, OR) was added to the U. maydis YEPSL cell culture and cells were incubated for 15 min at 25°C (Bortfeld et al., 2004 (link)). To analyze the U. maydis progression inside the maize plant, leaves samples from 3, 4 and 6 dpi infected plants were distained with ethanol, treated for 4 h at 60°C with 10% KOH, washed in phosphate buffer and then stained with propidium iodide (PI) to visualize plant tissues in red and wheat germ agglutinin (WGA)/AF488 to visualize the fungus in green. At least four leaves from two independent experiments were stained and visualized by fluorescence microscopy (Leica SPE DM2500, Leica, WZ, Germany). Image processing was carried out using the ImageJ software.