Eclipse te2000 e inverted microscope base

The localization of the eYFP-labelled Cre1-96 was determined by confocal microscopy and image processing using Fiji [43 (link)]. Samples were prepared from liquid cultures. Therefore, 10⁶ spores per mL of QM6acre1-96::eyfp were used to inoculate 20 mL of MA medium supplemented with 1% (w/v) d-glucose and 1% (w/v) lactose and incubated at 30 °C and 180 rpm for 16 h. A 10-μL sample was taken and embedded between two glass coverslips (24 × 60, 24 × 24). For the nuclear staining, 4 μL of a 1:10-diluted (distilled water) Hoechst 34580 stain (Thermo Scientific, 5 mg/mL in DMSO) was added before putting the glass coverslip on top of the sample and incubated for 10 min in darkness. Live-cell imaging was performed using a Nikon C1 confocal laser scanning unit sitting on top of a Nikon Eclipse TE2000-E inverted microscope base (Nikon Inc., Melville, New York, USA). An argon ion laser emitting a wavelength of 488 nm excited fluorescent proteins and Hoechst stained nuclei. The emission wavelength was detected with a photomultiplier in a range of 500–530 nm. Laser intensity and illumination time were kept the same for all samples. Pictures were taken as a single picture configuration at a resolution of 1024 × 1024 pixels.

To analyze the interfungal interaction by confocal microscopy, spores of T. reesei strain TUCIM 4817, carrying a gfp gene under the control of a histone 3A promoter, and Pestalotiopsis fici TUCIM 5788 were inoculated on two adjacent but separated PDA agar blocks mounted between the glass slides and the cover glass using a modified Riddell slide method [114] (Supporting information S3 Fig). The construct was incubated for 72 hours in a sterile wet chamber that hyphae of both fungi established the contacts. Live-cell imaging was performed using a Nikon C1 confocal laser scanning unit mounted on a Nikon Eclipse TE2000-E inverted microscope base (Nikon GmbH, Vienna, Austria). A Nikon Plan Apo VC 100×/1.4 with oil immersion objective lens was used. GFP was excited with an argon ion laser at 488 nm. The emitted fluorescence was separated by a Nikon MHX40500b/C100332 filter cube and detected with a photomultiplier tube within the range of 500–530 nm. Bright light images were captured simultaneously with a Nikon C1-TD transmitted light detector mounted behind the condenser turret.