Raman and FTIR imaging of lung tissue: Methodology for control samples

The paper describes Raman and FTIR images of normal lung tissue of an infant patient which is a prerequisite to understand the biochemical changes that accompany pediatric lung pathologies and malformations. A Raman image of 3420 spectra was recorded from a 5.7 mm × 6 mm tissue section in mapping mode using 785 nm excitation laser and a step size of 100 μm. A Fourier transform infrared (FTIR) image of 4096 spectra was recorded from the same sample at a lateral resolution of 63  μm using a spectrometer coupled to a focal plane array detector. Whereas the analysis of the FTIR image was complicated by dispersion artifacts, the analysis of the Raman image was impaired by intense spectral contributions of hemoglobin which overlap with spectral contributions of other proteins, lipids and nucleic acids. Data analysis procedures were optimized to reduce these effects. A multiplicative signal correction was performed to normalize the spectra within each dataset. Then, pseudo color images were constructed using cluster analyses. The most significant variations within both images were assigned to different concentrations of red blood cells. Furthermore, mucus and blood vessels were identified in the vibrational spectroscopic images. The different sensitivity of Raman and FTIR imaging for lung tissue constituents supports the combined use of both modalities.