Las x 2

The samples were mounted on microscope slides, and images were captured using a Leica TCS SP8 microscope (Wetzlar, Germany) with the HC PL APO CS2 63x/1.4 oil objective at the Laboratory of Microscopic Imaging and Specialized Biological Techniques University of Lodz. The 488 nm laser was used to excite the fluorescence, and the emission was collected by a hybrid detector in the range of 505–550 nm. To visualize the cells, the PMT transmission channel was used. LAS X 2.0.2.15022 software (Leica Microsystems, Wetzlar, Germany) was used to analyze the data. All settings were held constant throughout the experiments. All signals obtained from confocal microscopy were validated with profile view image analysis, and the diagrams presenting intensity values were placed under each microphotograph. The mean fluorescence intensity (expressed in arbitrary units (AU)) was calculated for each of the samples. The calculations were performed for at least 40 different points randomly selected in compartments with receptor expression.

Cells of C. albicans ATCC 10321 that were treated with essential oils were examined using the Leica TCS SP8 Confocal Laser Scanning Microscope (Leica Microsystems, Wetzlar, Germany) and the LAS X 2.0.2.15022 software (Leica Microsystems, Wetzlar, Germany) in the Laboratory of Microscopic Imaging and Specialized Biological Techniques, University of Łódź. The fluorescence excitation of oils was induced using UV (Leica Microsystems, Wetzlar, Germany) diode of 405 nm., while the detection was recorded by three separate detectors with 800 ± 5 gain and -0.5 offset: PMT 1 at a 430–480 nm (Blue Channel), PMT 3 at a 500–550 nm (Green Channel), and PMT 5 at a 600–650 nm (Red Channel). Moreover, a PMT Trans Channel was used to visualize the cells in transmitted light. Each sample was scanned in the ‘xyz’ axes to a depth of 30 µm (three-dimensional (3D) scan) while using HC PL APO CS2 63×/1.40 (Leica Microsystems, Wetzlar, Germany) Oil objective. Line Average of 4× was used to improve the quality of images. The images were visualised in 3D view using a surface and blend mode with an adjusted threshold for a cross-section of cells.

Confocal imaging of thrombi was performed with a Leica TCS SP8 confocal microscope with LAS X 2.0.2.15022 software (Leica Microsystems, Wetzlar, Germany) using the objective HC PL IR APO 40×/1.10 (water immersion). The 488 nm supercontinuum white light laser (WLL) (12% intensity) was used to excite the fluorescein-stained thrombi. The emission was collected by a photomultiplier tube detector in the range of 492–564 nm. Confocal Z-stack scans were performed at a rate of 400 Hz, zoom 1.0, pinhole 1.0 and line averaging set at 3 to improve image quality. In each field of a view 70 focal planes were acquired (logical size format X/Y/Z 512/512/70) [54 (link)]. For the analysis of images, FIJI software was used [55 (link)], according to our previously established protocol [56 (link)]. Briefly, the thresholding procedure was performed with the use of ‘Auto Local Threshold’ function (Bernsen method) with a radius value set at 5. To quantify the identified objects, the ‘3D Object Counter’ tool was applied, with a threshold set at 255, and a cut-off set at 20 µm³ (to exclude the objects too small to qualify them as thrombi). Volumes of separated thrombi were acquired for further analysis, and subsequently summarized to obtain total clot volume per sample.