# Raman Spectroscopy of Individual Cervical Exfoliated Cells in Premalignant and Malignant Lesions
## 4 Discussion  
When comparing the spectra of cervical samples of healthy patients with those of patients with cervical lesions, we observed differences in some bands, which mainly involve an increased intensity in the spectra for CIN patients. Furthermore, the increase in Raman spectroscopy band intensity was proportional to the degree of lesion present, the intensity being greater in CIN2 and CIN3/CIS lesions than in CIN1. In the literature, similar differences have already been reported in the Raman spectra of samples from healthy and cervical cancer patients, with increased intensities in the latter; most studies have focused on cell lines such as CaSki or HeLa or have used cell pellets instead of individual cells [17,18,31]. In the present study, bands that mark differences between samples of healthy patients and patients with a cervical lesion are those that correspond to lipid and protein cellular components (1077, 1396, 1440, 1546, 1603, 1650 cm\({}^{-1}\)) and nucleic acids (861, 1105, 1323, 1600 cm\({}^{-1}\)). Our Raman results were spectrally very similar in the cytoplasm and the nucleus. Previous studies reported Raman fingerprints for healthy cervical cells [13,28]. In a study conducted by [PERSON] et al. [19] in spectra from a high-grade lesion, a higher signal was obtained for nucleic acids (1485 and 1580 cm\({}^{-1}\)) in the nucleus and nucleic acids and proteins (1450 and 1650 cm\({}^{-1}\)) in the cytoplasm. In our study, the Raman spectra analyzed derive from the cytoplasm and nucleus of individual cervical exfoliated cells of the squamous epithelium's intermediate and superficial stratum. The superficial cells are 40-60 \(\upmu\)m in diameter and are flattened, thin, and generally polygonal in outline; for its part, the nucleus is circular, small (\(\sim\)6 \(\upmu\)m), and located in the center of the cell. On the other hand, the intermediate cells are of very similar size or a little smaller than the superficial cells; their shape is more rounded, cytoplasm  
Figure 4: Raman spectra of cervical cells of healthy patients with varying degrees of lesion. (**A**) Spectra corresponding to the cytoplasm and (**B**) Raman spectra obtained from the nucleus. Spectra of different groups: Healthy (black), viral latency (red), CIN1 (blue), CIN2 (pink), and CIN3/CIS (green). Each color corresponds to the cellular components represented in the box by the graphs. slightly thicker than that of the superficial cells; the nucleus is central, rounded, and its size is \(\sim\)8 \(\upmu\)m. In the presence of any premalignant lesion (CIN), cellular changes occur that vary depending on the degree of lesion present, being more evident and to a greater extent in high-grade lesions; some of the changes are nuclear increase that results in an alteration of the nucleus/cytoplasm ratio, anisonucleosis, or irregular nuclear contour [32]. The intermediate and superficial cells are in a state of differentiation compared to parabasal and basal cells. The lower layers of the squamous epithelium are stem cells where HPV entry occurs and the infection begins. These cells are in the process of maturation, where the highest expression of HPV oncogenes E6/E7 and other late HPV [33] genes occur. The E6/E7 proteins inhibit critical cellular processes of DNA damage repair or its regulation by interfering with the functions of tumor suppressor genes such as pRB through E7 activity or by degrading proteins that activate signaling cascades involved in the repair of damaged DNA before the synthesis or replication of higher amounts of mutated genetic material, via the effects of E6 on p53 [34,35]. In addition to the higher expression of E6/E7 genes in the intermediate and superficial cells, an increased expression of late genes also occurs, such for the genes encoding L1 and L2 proteins, which form the viral capsid [36,37]. Therefore, there is an increase in the transcription, translation, and assembly of viral proteins in these cells.