Oil immersion objective c apochromat63

Image acquisition was done on an inverted confocal laser scanning microscope LSM 780 (Carl Zeiss AG) using the ZEISS oil immersion objective (C-Apochromat63) and the ZEN 2011 (black version) software (Carl Zeiss AG). All images were displayed as three-dimensional (3D) z-stacks (13 stacks with an interval of 1 µm; frame sizes of 1250 × 1015 pixels). Z-stacking was used to generate a 3D representation to understand the overall cell structures. Thus, a false interpretation due to different observation levels (confocal principle) could be avoided. The cell areas for n = 50 cells per sample out of n = 2 experiments with ImageJ software (Wayne Rasband, National Institute of Health) using the LSM images from the actin cytoskeleton staining.

MG-63 cells were cultured for 30 min (50,000 cells/cm²) and 24 h (30,000 cells/cm²) on Ti, Ti-Col I, and Ti-TMS-PEI, respectively, and on Ti-G20, Ti-G20-Col I, and Ti-G20-TMS-PEI, respectively. Cells were then washed with PBS (Sigma-Aldrich, Germany), fixed with 4% PFA for 10 min at room temperature (RT), rewashed with PBS, and permeabilized with 0.1% Triton X-100 (Merck, Germany) for 10 min at RT. After another washing step with PBS, the cells were incubated with phalloidin-tetramethylrhodamine B isothiocyanate (phalloidin-TRITC, diluted 1:15 in PBS, Sigma-Aldrich) for 30 min at RT in the dark. Cells were again washed with PBS, embedded with DAPI-containing Fluoroshield™ and analyzed with the inverted confocal microscope LSM 780 (Carl Zeiss) using the ZEISS oil immersion objective (C-Apochromat 63) and the ZEN 2011 (black version) software (Carl Zeiss). To visualize cells’ actin cytoskeleton on Ti-G20 chips, confocal microscopy with three-dimensional (3D) z-stack generation was applied.