Axio observer spinning disc confocal microscope

All microscopy experiments were performed on an Axio Observer Spinning Disc Confocal Microscope (Carl Zeiss, Jena, Germany) with 63×/1.2 Oil or 100×/1.4 Oil objectives. 405 nm, 488 nm, and 561 nm laser lines were used to excite the fluorescent proteins. Fungal conidia were grown overnight at 37°C in AMM as adherent cultures in ibidi well chamber slide (ibidi, Gräfelfing, Germany). Time-lapse microscopy was performed with a modified, previously described protocol (30 (link)). The culture was incubated at room temperature during the experiment, lasting for two to three hours. When necessary, fixation of fungal hyphae was achieved with a final concentration of 4% formaldehyde (AppliChem, Darmstadt, Germany). For the A. niger, the expression of strains was induced using up to 20 µg/mL final concentration of doxycycline.

FRAP experiments were performed as described elsewhere with an iMIC digital microscope with a 60× objective (Till Photonics) at RT (Phair et al, 2004 (link)). Pictures were acquired with a series of five z-slices each separated by 0.5 μM. Four images were taken before the ROI was bleached with 100% laser power, a dwell time of 1.2 s/μm², a line overlap of 42%, and an experimental loop count of 10–20. Pictures were taken at a constant time interval of 0.9 s after bleaching except SH3_Boi1-GFP where signal recovery was measured each 0.25 s in a single z-layer. Initial z-slice projection and fluorescence quantification was performed with the software iMIC Offline analysis. Alternatively, an Axio Observer spinning-disc confocal microscope (Zeiss), equipped with a Zeiss Plan-Apochromat 63 Å∼/1.4 oil DIC objective, a 488-nm diode lasers, and an UGA-42 photo-manipulation system was used (Rapp OptoElectronic). Initial signal measurements were ImageJ based. Subsequently, all data sets were double-normalized using Excel. The software Prism 6.0 (GraphPad) was used for the fitting of the double-normalized data to a one-phase association curve.