Spectrum spotlight 400

Prior to data acquisition, a chemical deparaffinization step was performed using octane with a purity of ≥ 98.0% (Sigma Aldrich, Buchs, Switzerland) for 4 hours at a temperature of 40°C and dried afterwards as described by Pallua et al [36 (link)]. All MIR images were recorded in transmission mode using a commercially available infrared microscope Spectrum Spotlight 400 (Perkin-Elmer, Massachusetts, USA). Spectral data were recorded using the software “Spectrum IMAGE R1.8.0.0410” with a spectral resolution of 4 cm^-1 and a pixel size of 25 μm. Before each measurement, a background spectrum was recorded outside the sample area to calculate a ratio[36 (link)]. Each final spectrum consisted of two co-added scans. The result of the MIR imaging measurement was a hyperspectral cube composed of both a local and a spectral information. The measurement time per sample strongly depends on the size of the sample and the used pixel size on the instrument. The average time for scanning one slide was 30 minutes. Each recorded pixel therefore had an associated MIR spectrum representing the local chemical information. By superimposing the previously gained information from the HE stained histologic slide with the recorded spectra, the development of a multivariate discrimination model could be achieved.