Isys software

Cell cultures of 7F2 osteoblasts were analyzed by FTIR imaging in a PerkinElmer Spotlight 400 spectrometer, with 6.25 μm spatial resolution, 4 cm⁻¹ spectral resolution and 8 co-added scans. Intact micromasses on Si wafers were imaged in transmittance mode and analyzed using ISys software (Malvern Instruments), to obtain the distribution images of the mineral and protein components based on the area of the ν₃ PO₄ and amide I bands, respectively. The spatial correlation analysis between mineral and protein distribution was done using ImageJ software (National Institutes of Health). Briefly, pairs of grayscale images were analyzed with the “colocalization threshold” plugin, to quantify the pixel intensity spatial correlations of components from both images. The Pearson correlation coefficient (R) and percent areas of correlation were obtained as the quantitative outcomes of the analysis. To build overlay images of apatite and ACP, grayscale images were obtained based on the positive values of the inverted second derivative peaks at ~1015 cm⁻¹ and ~992 cm⁻¹, peaks unique to apatite and ACP, respectively, using ISys software. Corresponding images were set to red and green channels and merged using ImageJ software.