Digital microscope vhx 500f

Microscopic analysis was performed using an Axio Imager.M2 equipped with an AxioCam MRm camera (ZEISS) or an Axio Imager.M1 (ZEISS) equipped with a CoolSNAP HQ2 CCD camera (PHOTOMETRICS). All images were processed with ZEN 2.3 blue edition (ZEISS).
Chlorazol Black E staining was performed according to [104 (link)]. For microscopic analysis of cells after TM treatment U. maydis strains were grown in CM to an OD₆₀₀ of 0.35. TM was added to a final concentration of 5 μg/ml and cells were incubated for the indicated time to induce the UPR.
For detection of reactive oxygen species (ROS) in infected leaf tissue, 3,3’-diaminobenzidine (DAB) was used as described previously [61 (link)]. Briefly, leaves (third leaf) were detached with a razor blade 1 cm above and 2 cm below the injection site 24h post infection and incubated for 12h in 1 mg/ml DAB solution under darkness at room temperature. For decolorization, leaves were immersed in ethanol (96%) for 48h. For storage of the specimens, the leaves were transferred into 10% (v/v) glycerol. Brown polymerization products resulting from the reaction of DAB with ROS were microscopically identified using a binocular microscope (Keyence Digital Microscope VHX-500F).

The construction of the two-hybrid plasmids used in this study and verification of the expression of the GAL4 fusion proteins is described in Method B S1 File.
S. cerevisiae strains AH109 and Y187 were used for yeast-two hybrid experiments. Mat α strain Y187 was transformed with bait plasmids pGBKT7, pBD-Smatg8, pBD-Smatg12, pBD-Smatg3 or pBD-Smatg7 and transformants were selected for tryptophan prototrophy, whereas Mat a strain AH109 was transformed with prey plasmids pGADT7, pAD-ranBPM, pAD-Smatg8, pAD-Smatg12, pAD-Smatg3 or pAD-Smatg7 and transformants were selected for leucine prototrophy. Recombinant AH109 and Y187 strains were mated and selected on solid SD minimal medium lacking both, tryptophan and leucine. Alternatively, both plasmids were co-transformed into strain AH109. Interaction of the bait and prey fusion constructs was confirmed by growth on selective SD minimal medium lacking histidine and adenine. Cells grown to the log phase in liquid SD minimal medium without leucine and tryptophan were diluted to an optical density (OD) of 0.1. From this main dilution, 20 μl were spotted in a 1:10 dilution series onto selective SD plates, which were incubated at 30°C for five days. Yeast growth was visualized using a “Digital Microscope VHX-500F” (Keyence, Germany).

The morphology of the produced particles was investigated using a scanning electron microscopy (JEOL JSM-7600F, JEOL Europe bv, Zaventem, Belgium) at low voltage (2 kV) and working distance of 8 mm. Samples were sputtered shortly with gold using a BAL-TEC SCD 005 Sputter Coater (Bal-tec GmbH, Wetter, Germany) at 25 mA. In some cases optical microscopy (Keyence digital microscope VHX-500F, Keyence International, Mechelen, Belgium) was used to determine size and shape of the particles. Obtained micrographs were then analysed using the software program Image J and further investigated using the statistical program SPSS.

The morphology of the produced particles was investigated using a scanning electron microscopy (JEOL JSM-7600F, JEOL Europe bv, Zaventem, Belgium) at low voltage (2 kV) and working distance of 8 mm. Samples were sputtered shortly with gold using a sputter coater (SCD 005, BAL-TEC, Wallruf, Germany) at 25 mA. In some cases, optical microscopy (Keyence digital microscope VHX-500F, Keyence, Mechelen, Belgium) was used to determine size and shape of the particles. Obtained micrographs were then analyzed using the software program Image J and further investigated using the statistical program SPSS.

For the investigation of sexual structures and hyphae, S. macrospora strains were grown for two to ten days on SWG covered glass slides and documented with a VHX-500F Digital Microscope (Keyence, Germany) or AxioImager M1 microscope (Zeiss, Jena, Germany) with differential interference-contrast (DIC). Images were captured using a Photometrix CoolSNAP HQ camera (Roper Scientific, Photometrics, Tucson, AZ, USA). Image processing was done using ZEISS ZEN Digital Imaging (version 2.3; Zeiss).
For fluorescence microscopic analysis, S. macrospora strains were grown on solid medium on top of a piece of cellophane (2 cm × 2 cm) in petri dishes at 27 °C for the indicated hours or days. For the detection of the eGFP signal chroma filter set 49002 and for DsRED/TagRFP-T chroma filter set 49005 (exciter ET470/40x, ET545/30x, emitter ET525/50 m, ET620/60 m and beamsplitter T495lpxr, T570lp) were used.

To define growth behavior and sexual development, S. macrospora strains were grown on solid SWG medium over 10 d and documented with a VHX-500F Digital Microscope (Keyence, Osaka, Japan). For growth-rate determination, six replicates each of S. macrospora wt strain, Sm::Smarp1-TagRFP-T and Sm::Smarp1-mNG, were grown in 30-cm race tubes filled with solid SWG medium over 7 d under continuous light at 27 °C. After 3 d, the growth front was marked every day at the same time to determine the growth velocity in cm/day.
For the determination of the C. graminicola growth rate, defined mycelial plugs of CgM2, CgM2::Arp1-TagRFP-T, and CgM2::SmArp1-mNG from pre-cultures were transferred to fresh CM plates without selection. After 3 d, the growth area was recorded using an Epson Perfection V600 Photo scanner in four subsequent days. The optical evaluation of the growth area was done using the measuring tool of Fiji [71 (link)] on scaled images. The growth rate of four replicates was determined by the difference of the growth area of two subsequent days in cm².

To investigate vegetative hyphae and sexual structures, S. macrospora strains were grown on solid SWG medium covered with a piece of cellophane (0.5 cm × 0.5 cm) over 2 to 9 days and documented with an AxioImage M1 microscope (Zeiss, Jena, Germany) using differential-interference contrast (DIC) or a VHX-500F Digital Microscope (Keyence, Neu-Isenburg, Germany). Images were captured with a Photometrix CoolSNAP HQ camera (Roper Scientific, Photometrics, Tucson, AZ, USA). Image processing was done using ZEISS ZEN Digital Imaging (version 2.3; Zeiss, Jena, Germany) and the Affinity Publisher software (version 1.9.2.1035, Serif (Europe) Ltd., Nottingham, UK, https://affinity.serif.com/de/publisher/; accessed on 1 March 2021).
For fluorescence microscopic analyses, S. macrospora strains were grown on selective BMM medium on top of cellophane sheets or on BMM-covered glass slides for 24 h at 27 °C. For the detection of the EGFP signal Chroma filter set 49002 (exciter ET470/40x, ET525/50m, beamsplitter T495lpxr) and for TagRFP-T/tdTomato Chroma filter set 49005 (exciter ET545/30x, emitter ET620/60m and beamsplitter T570LP) was used.