Micro ATR-FTIR spectroscopic imaging of colon biopsies with a large area Ge crystal

A new large-area germanium ATR crystal is utilised with an FTIR microscope to improve the acquired images of de-paraffinized colon biopsy sections, without recourse to a synchrotron source. The large crystal (⌀ = 28 mm) offers significant improvements compared to slide-on small germanium crystal (⌀ = 3.5 mm); for example, it facilitates more uniform distribution of higher signal intensity within the field of view and more rapid acquisition time. Mapping of a larger sample area up to ca. 350 × 350 μm² with this new set-up, coupled with imaging using an FPA detector, is demonstrated for the first time on biological specimens. The performance of k-means clustering algorithm applied to classify the different anatomical structures of the colon biopsies is greatly improved with mapping. Comparison of H&E stained adjacent tissue sections with false-colour k-means images strongly support differentiation of five distinct regions within tissues. The efficiency of the methodology to categorise colon tissues at various stages of malignancy is analysed via multivariate chemometrics. The second derivative spectra extracted from the crypt region of the colon were subjected to Partial Least Squares classification. Good separation between data in clusters occurs when projecting spectra on a PLS score plot on a plane made by the first 3 principal components. Important spectral biomarkers for colon malignancy classification were identified to exist mostly in the fingerprint region of the FTIR spectrum based on the chemometrics analysis.