FTIR spectral imaging was applied on formalin-fixed paraffin-embedded biopsies from colon and skin cancerous lesions. These samples were deposited onto different substrates (zinc selenide and calcium fluoride respectively) and embedded using two types of paraffin. Formalin fixation followed by paraffin embedding is the gold standard in tissue storage. It can preserve molecular structures and it is compatible with immunohistochemistry. However, paraffin absorption bands are significant in the mid-infrared region and can mask some molecular vibrations of the tissue. Direct data processing was applied on spectral images without any chemical dewaxing of the tissues. Extended Multiplicative Signal Correction was used to correct the spectral contribution from paraffin. For this purpose, the signal of paraffin was modelled using Principal Component Analysis and paraffin spectra were removed from the raw images based on an outlier detection. Then, pseudo-colour images were computed by K-means clustering in order to highlight histological structures of interest. This robust chemometrics methodology was applied on the two samples. Tumour areas were successfully demarcated from the rest of the tissue in both colon and skin independently of the embedding material and of the substrate.

Raman spectroscopy represents an analytical, non-destructive, and dynamic method to evaluate the permeation of actives in the skin layers.