Em cpd3000

For observation of the cells under SEM, the cells were first fixed after 6 h seeding on the PDMS platform, which was described in ‘Immunofluorescence staining and confocal imaging’ section. The cells were rinsed with PBS at 37°C, followed by fixation with 4% PFA for 15 min. After thoroughly rinsing with PBS, the PDMS platforms were dehydrated through an ascending concentration of ethanols (30, 50, 70, 80, 90, 95, and 100%). To reduce the artifacts created by surface tension, the cells were supercritically dried using a critical point dryer (EM CPD3000, Leica, Hesse, Germany). The PDMS platforms were then sputter-coated with gold.

To investigate the effect of 2d compound on T. rubrum CBS cells, scanning electron microscopy was applied. The healthy volunteer nails were cut into small pieces of 2.5 × 3.5 mm² and sterilized at 121 °C for 15 min. The sterilized nail fragments were exposed to a spore suspension containing 2–5 × 10⁵ CFU/mL for 2 h at 28 °C. The nails were then removed from the spore suspension and placed in a 24-well plate containing MM-Cove medium⁸⁶ . 2d solution was added at concentrations 0.5 × MIC (16 mg/L), 1 × MIC (32 mg/L), and 2 × MIC (64 mg/L). After 24 h of exposure to 2d, the nail fragments were fixed with 2.5% glutaraldehyde and dehydrated in a graded ethanol series. Then, the samples were placed into a critical point dryer (Leica EM CPD 3000) and were coated with ionized gold (Leica EM ACE 200). The nail fragments were observed in the Phenom ProX Scanning Electron Microscope belonging to the Department of Invertebrate Zoology and Hydrobiology, University of Lodz. The SEM method was carried out following relevant guidelines and regulations. The study was approved by the institutional ethics committee (the Local Ethics Committee in Lodz, Poland). Written informed consent was obtained from the volunteer.

The cell samples cultured to specific time points were extracted and fixed using 4% paraformaldehyde. Subsequently, they underwent three rounds of PBS cleaning and visualized using a confocal laser microscope (HR Confocal Laser Scanning Microscope, Nikon, A1). The Z‐stack imaging covered a range from 0 to 1000 µm, with intervals of 10 µm. The fixed sample was also subjected to a gradient alcohol dehydration process followed by treatment with a critical point dryer (Leica, EM CPD3000) to obtain a wholly dried sample. Subsequently, the dried samples were subjected to sputtering techniques, and the cellular morphology of each sample was examined using scanning electron microscopy at a 5 kV acceleration voltage.