Ultraview ers spinning disk confocal system

Conidiospores were inoculated in 35 mm glass-bottom microwell dishes (MatTech) containing minimal media with glucose as the carbon source and urea as the nitrogen source. Imaging was carried out at room temperature using an Ultraview ERS spinning disk confocal system (Perkin-Elmer) fitted with an Orca-AG camera (Hamamatsu) on a TE2000-U inverted microscope (Nikon) with a 60× 1.40 NA Plan Apochromatic objective (Nikon). For some experiments, imaging was with an Ultraview Vox spinning disk confocal system (Perkin-Elmer) fitted with dual C9100-13 cameras (Hamamatsu) run by Volocity software (Perkin-Elmer). Data are displayed as maximal intensity profiles. Benomyl (Sigma) was used at a concentration of 2.4 µg/ml which is sufficient to depolymerize all microtubules [131] (link). Image analysis, quantification, pixel line intensity profiles and kymograph generation was carried out using ImageJ freeware (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997–2008). To quantify histone H1 fluorescence levels the average pixel intensity less the background was determined for an identical sized area for each cell. Each time series was aligned for mitotic entry and values normalized relative to the last frame in G2 before mitotic entry which was set to 100%. Data points represent the mean +/− standard deviation.

AnTat1.1E AMLuc/tdTomato parasites were monitored in the ear of Kdr (Flk1) C57BL/6J Rj mice by spinning-disk confocal microscopy as described previously (Rotureau et al., 2012 (link)). Briefly, mice were first anaesthetised by IP injection of ketamine (Imalgene 1000 at 125 mg/kg) and xylazine (Rompun 2% at 12.5 mg/kg). Mice were wrapped in a heating blanket and placed on an aluminium platform with a central round opening of 21 mm in diameter. A coverslip was taped on the central hole and the mouse was positioned so that the ear was lying on this oiled coverslip. Imaging was performed using an UltraView ERS spinning-disk confocal system (Perkin Elmer) with a x40 oil objective (1.3 numerical aperture). Movies were acquired by an EM-CCD camera (Hamamatsu) controlled by the Volocity software (Perkin Elmer) with an exposure time of 500 ms for a total of 30 to 120 s. Images were analysed using ImageJ 1.48v and its plugin Bio-formats importer (NIH).