Microscopic Imaging of Fungal Hyphae

Spores were cultured at 10⁴ spores/ml in 0.2 ml of liquid glucose minimal medium for 10 h at 37°C with 5% CO₂ in the dark on MatTek dishes (MatTek, P35G-1.0-14-C). At this point, hyphae of various sizes had formed. Hyphae were imaged unfixed on an Andor W1 spinning disk confocal with a Nikon Eclipse Ti inverted microscope equipped with a CFI Plan Fluor 100× Oil objective (Nikon). 3D rendering and image analysis were performed in Nikon Elements Viewer (Nikon). For FM4-64 staining, hyphae were incubated in 10 μM FM4-64 in phosphate-buffered saline (PBS) for 15 min on ice and then briefly incubated in 37°C for 5 min before being washed twice with PBS and imaged as described above. Detection of B-glucan using Dectin-1 binding protocols was performed as previously published (72 (link)). Briefly, 12-h hyphal cultures were blocked in fluorescence-activated cell sorting buffer containing fetal bovine serum for 30 min, washed twice with PBS, and then incubated in 150 μl of soluble Dectin-1 for 1 h at room temperature. Hyphae were stained with goat anti-human IgG–Alexa Fluor 594 in PBS for 1 h at room temperature.