Raman Micro Spectroscopy for the Characterisation of Cervical Cancer.

The primary purpose of this study is to evaluate the potential of FTIR and Confocal Raman micro spectroscopy (CRM) in elucidating the biochemical changes occurring in different layers of the cervical epithelium including basal, superficial and the underlying connective tissue known as stroma during the progression of Cervical Intraepithelial Neoplasia (CIN) to cancer. Initially the two techniques were compared and Raman was chosen based on its higher spatial and spectral resolution. The sample preparation and spectral measurement procedures were optimised and all samples were formalin fixed paraffin processed, dewaxed using xylene, and measured on calcium fluoride windows. Raman spectra were recorded using a source wavelength of 785nm and a X100 dry objective lens. Raman micro spectroscopy was able to differentiate the normal region of the cervical tissue sample into three layers including stroma, basal/para-basal and superficial layers on the basis of the spectral features of the collagen, DNA bases and glycogen as well as discrimination of the diseased areas from the normal areas. In particular Raman spectroscopy could describe the biochemical changes in the diseased samples in detail. On moving from normal to abnormal regions of the cervical tissue sample, the characteristic Raman features of the basal layer were observed in the superficial layer and in the stroma. Notably, the normal region of a CIN III sample was found to have biochemical information similar to the abnormal region. This has been indicated by the absence of the collagen Raman spectral bands in the stromal layer as well as absence or minute presence of the glycogen bands in the superficial layer. A comparison of the Principal Components Analysis (PCA) loadings of the HPV negative and positive cell lines (C33A and CaSki), with those of the basal layers of normal and abnormal tissue samples showed no strongly matching Raman signatures which could lead to the identification of signatures of HPV infection in the cervical cancer tissue samples. 3 However, a feature associated with the amide-III beta sheet (1222 cm-1) was found to be consistently present in the PCA loadings contributed by the basal layers of the intermediate and abnormal samples. This was much reduced in intensity in the most extreme abnormal sample and the carcinoma in situ samples. This feature may be considered as an early marker of disease progression, but further investigation is needed to confirm this finding. KMCA indicated the possibility of the migration of basal cells into the superficial and stromal layers and subsequent PCA led to the conclusion that basal cells indicated by high DNA content and lack of glycogen are progressing to the superficial layer due to the progression of the disease. In the case of stroma, the basal like characteristics are actually associated with the biochemical changes in the stromal cells and there is no migration of these cells into the stroma. In addition, during cervical cancer progression, relative to the DNA, collagen has a diminished contribution at some points in the Raman map of the stroma and KMCA recognised the greater similarities with the DNA rich cells of the basal layer. This has been supported by the enhanced expression of p16 protein in the basal and superficial layers rather than in the stroma