Dmi 6000 b inverted microscope

Aspergillus nidulans conidiospores were inoculated in uncoated glass-bottom µ-dishes (Ibidi GmbH) containing 2.5 mL watch minimal medium (WMM^{57 (link)}) supplemented with 25 mM NaH₂PO₄, 5 mM ammonium (+)-tartrate, and 0.5% glucose. After 16 h at 25 °C medium was replaced with freshly prepared WMM supplemented with 100 mM Na₂HPO_4. Samples were cultured for an additional 1.5 h at 37 °C prior to observation of fluorescence (see text for details of enaA and enaB induction).
Differential Interference Contrast (Normarski optics) and fluorescence images were acquired from in vivo cultures with a Leica DMI-6000b inverted microscope coupled to an ORCA-ER digital camera (Hamamatsu Photonics) and equipped with a 63 Plan Apochromat 1.4 N.A. oil immersion objective (Leica) and a GFP filter (excitation 470 nm; emission 525 nm). Images were acquired using Metamorph (Molecular Dynamics) software and processed using ImageJ free-software (https://imagej.nih.gov/ij).

C. albicans vacuole integrity was assessed using the lipophilic vacuole membrane dye MDY-64 (Molecular probes, Fisher Scientific) following the manufacturer's recommended procedure. Briefly, cells were grown overnight on RPMI liquid medium with pH 4.5 at 30°C. Cells were pelleted and washed twice with fresh RPMI pH 4.5 and resuspended in the same medium at an OD₅₉₅ of 0.1. VPA was added at different concentrations (10, 50, and 100 μg/ml). Cells were incubated for 2 h at 30°C under agitation. Aliquots were taken from VPA-treated and non-treated cultures and the MDY-64 was added at a final concentration of 10 μM. Cells were incubated at room temperature for 3 min prior to confocal microscopy visualization. Images were acquired with a 1.3-numerical-aperture (NA) 63x objective on a Leica DMI6000B inverted microscope connected to a Hamamatsu C9100-13 camera.
Pan1-green fluorescent protein (GFP), End3-GFP and LIFEACT-GFP (Epp et al., 2013 (link)) were visualized using confocal microscopy as follow: an overnight culture was diluted in SC supplemented with 10 or 50 μg/ml VPA to an OD_595nm of 0.05 and grown for four generations at 30°C under agitation. Cells were imaged as described for the vacuole staining experiments.