Axiocam 208

The effect of the EO on the ability of C. albicans ATCC 10231 to form hyphae was determined in 96-well microtiter plates. Briefly, the cell suspension was diluted to 5–10 × 10⁶ CFU/mL in 200 µL of RPMI 1640 supplemented with 10% fetal bovine serum (FBS, Invitrogen) that contained different concentrations (47–375 µg/mL) of EO or 1% DMSO. The plate was incubated at 37 ± 2 °C with agitation (200 rpm) for 5 h and visualized using 40× lens brightfield Zeiss microscope. The photographs were taken with a Zeiss Axiocam 208 color camera [57 (link),58 (link)].

Temporary slides were prepared by means of trapping spores in a drop of water on a glass slide with a coverslip, which was secured with glyceel. The size of the conidia was determined, measuring both the length and the width of 30 spores, using a Zeiss Axiolab 5 light microscope equipped with an Axiocam 208 camera. The scanning electron microscope preparation of spores of different Metarhizium species, including M. majus, M. robertsii (GenBank accession number MT378171), M. pinghaense (MT895630), and M. brunneum (MT380848), was undertaken and photographed by the Central Analytical Facility of Stellenbosch University. The morphological identification of the entomopathogenic fungi was done according to Humber’s key [14 (link)].

All Fusarium isolates were incubated on PDA plate in the dark at 25 °C for 7 days. Colony color and colony texture were observed for each isolate. To determine the size of well-developed macroconidia (n = 30) and the number of septa, these Fusarium isolates were incubated on PDA plates at 25°C for 7 days with light/dark cycle of 8/16 h. The macroconidia were observed under light microscopy (Zeiss Axiolab5 equipped with an Axiocam 208 color industrial digital camera).

The microscopic investigations were performed using an inverted microscope ZEISS Axio Vert.A1 (Zeiss, Shanghai, China). The studies were performed with the objective magnifications LD A-Planx40/0,55 ph1 (air), A-Planx100/1,25 Oil Ph2 oil, and the color camera Axiocam 208 (Zeiss, Shanghai, China). For the imaging, the emulsions were inserted into a 1 μ-Slide VI^0,1 cuvette (ibidi GmbH, Gräfelfing, Germany). For the presentation, the resolution of the images were automatically adjusted by the best fit ZEN2.5 software (Zeiss, Jena, Germany).

The microscopic studies were performed using an inverted microscope ZEISS Axio Vert.A1 (Zeiss, Shanghai, China) equipped with a color camera Axiocam 208 (Zeiss, China). Imaging was performed using two kinds of objectives with different magnifications: LD A-Plan (×40/0.55; phase 1 (air)) and A-Plan (×100/1.25; phase 2 (oil)). For the imaging, the emulsions were inserted into a 1 μ-Slide VI 0.1 cuvette (ibidi GmbH, Gräfelfing, Germany). For the presentation, the resolution of the images was automatically adjusted by the best fit with the ZEN2.5 software (Zeiss, Jena, Germany).

Cells were seeded in 60 mm diameter plastic culture dishes with the test materials, control material, and without the material and incubated under cell culture conditions. At T1, T2, and T3 follow-ups, the dishes were observed using a phase-contrast light microscope (ZEISS Primovert, Jena, Germany), and a ZEISS Axiocam 208 colour camera was used to capture the images at 10× and 20×.

The right lung fragments of the mice were fixed in 4% paraformaldehyde and then embedded in paraffin. Subsequently, sections 4 μm thick were cut and stained with hematoxylin and eosin (H&E) or Masson’s trichrome staining to be analyzed by light microscopy (Primo Star 3, Carl Zeiss, Jena, Germany) coupled with an Axiocam 208 color camera and ZEN Microscopy Software, version 3.9 (Carl Zeiss, Jena, Germany). H&E-stained slides were captured at 10× magnification and used to evaluate the magnitude of the alveolar spaces (degree of emphysema) by quantifying the mean linear intercept (Lm). For this, free areas of blood capillaries and respiratory ducts were considered, and a grid of 10 × 10 lines was placed over each field. At least three fields were considered per microscope slide. The Lm was calculated by dividing the total horizontal length of all the lines by the total number of alveolar septa that intercepted the grid lines [60 (link),61 (link)]. Masson’s trichrome staining was used to measure the peribronchial fibrosis in lung tissue under 10× magnification. Cross sections of small airways (<2 mm in diameter) were considered, and collagen deposition was calculated as the percentage of the area stained in blue in terms of the total area of the airway [62 (link)].