Nis elements software

The microstructures of the studied cement mortar mixtures were studied by scanning electron microscopy (SEM) by using a field emission gun scanning electron microscope Merlin-ZEISS (FEGSEM, Zeiss, Jena, Germany) equipped with a secondary electron detector operating at an acceleration voltage of 15 kV, probe current of 300–800 pA, and a working distance of 6–18 mm. The chemical compositions of the materials’ surfaces were determined by energy-dispersive X-ray spectroscopy (EDS). SEM images were analyzed using the NIS-Elements software (Laboratory Imaging Ltd., Prague, Czech Republic). The vacuum-dried tested samples were mounted on aluminum stubs using double-sided conductive carbon tape and sputter-coated with gold/palladium using a Quorum SC7620 sputter coater (Quorum Technologies Ltd., Lewes, United Kingdom).

Detection and quantification of mineralization was performed by Alizarin Red staining according to established procedures [24 (link)]. Briefly, after RPM exposure for 5 days, all cell samples were fixed with 4% paraformaldehyde for 15 min. Cells were washed with deionized H₂O before adding Alizarin Red solution (40 mm, pH 4.1) to each well. The plates were incubated at room temperature for 20 min with gentle shaking. After removal of excess dye, the wells were washed four times with abundant deionized H₂O and shaken for 5 min. The plates were left an angle for 2 min to facilitate removal of excess H₂O. Stained monolayers were visualized by a Nikon upright microscope ECLIPSE Ci-S (Nikon Corporation, Tokyo, Japan) connected to a Nikon digital camera. Images were acquired at 10× magnification using NIS-Elements software (5.30.01; Laboratory Imaging, Prague, Czech Republic).
For quantification of staining, a 10% acetic acid solution was added to each well, and the plate was incubated at room temperature for 30 min with shaking. The monolayer was then scraped from the plate with a cell scraper (Fisher Lifesciences, Waltham, MA, USA), transferred to a 1.5 mL tube and then centrifuged at 20,000 g for 15 min. Aliquots (150 μL) of the supernatant were read in triplicate at 405 nm in 96-well format using opaque-walled, transparent-bottomed plates (Fisher Lifesciences).

Detailed microscope observations of the nuclear structure were carried out with Nikon Eclipse 80i and Ni-U epifluorescence microscopes under × 100 and × 60 planachromatic immersion objectives. Fluorescence signals were visualized with the aid of two filter sets, each with a narrow-band excitation fitted precisely to the corresponding emission peak of the HBO lamp: (1) excitation 360–370 nm and emission 435–485 nm for DAPI; (2) excitation 430–440 nm and emission 470 nm for CMA₃. Extended-depth-of-focus (EDF) images were obtained by capturing 10–15 different focal planes of the same object by cooled monochrome DS-2MBWc or DSQi1 cameras (Nikon) both controlled by NIS Elements software (Laboratory Imaging, Ltd.). The frames were stacked and combined into one image using the EDF function.