Metamorph metafluor combination software package

To visualize the process of septation, conidia of indicated strains were incubated in indicated liquid medium on sterile glass coverslips, respectively, at related temperatures prior to observation under a microscope. The signal of septum was observed in live cells by placing the coverslips on a glass slide. Then removed the media from coverslips and washed three times by phosphate buffered saline (PBS). After that, 4% paraformaldehyde (Polyscience, Warrington, PA) was used to fix the cells and Calcofluor white (CFW) (Sigma-Aldrich, St. Louis, MO) was added for septum and chitin staining for keeping in dark for 10 min. After staining, the staining solution was removed and washed with PBS for three times. Finally, above coverslips were placed on a glass slide and sealed with nail oil. Microscopic images of cells were collected with a Zeiss Axio Imager A1 microscope (Zeiss, Jena, Germany). These images were then collected and analyzed with a Sensicam QE cooled digital camera system (Cooke Corporation, Germany) with the MetaMorph/MetaFluor combination software package (Universal Imaging, West Chester, PA), and the results were assembled in Adobe Photoshop 7.0 (Adobe, San Jose, CA). A similar approach was taken to visualize the localization of MobA-GFP in related strains. 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO) was used to stain DNA.

For hyphal microscopy, conidia were inoculated onto precleaned glass coverslips overlaid with MMPDR liquid media. The strains were grown on coverslips at 37°C for various durations prior to observation under a microscope. The DNA was stained using 4,6-diamidino-2-phenylindole (DAPI) after fixing the cells with 4% paraformaldehyde (Polyscience, Warrington, PA) (42 (link), 43 (link)). Differential interference contrast (DIC) images and fluorescent images of the cells were collected using a Zeiss Axio imager A1 microscope (Zeiss, Jena, Germany).
For observation of conidiophore structure, the slide culture method for microscopic observation was performed as previously described (31 (link), 44 (link)), with a few modifications. Conidia were inoculated on the edge of a small square of agar medium placed on top of a coverslip, which was placed in a petri dish containing solidified agar to keep it moist. Another coverslip was placed on top of the agar square after inoculation. The coverslips with aerial hyphae and attached conidiophores were imaged using a SensiCam QE cooled digital camera system (Cooke Corporation, Germany) and analyzed with the MetaMorph/MetaFluor combination software package (Universal Imaging, West Chester, PA).