# Raman Spectroscopy of Individual Cervical Exfoliated Cells in Premalignant and Malignant Lesions
The sample material is analyzed without special preparations or modification along the surface on which the analysis is carried out [13]. This technique is based on measuring the light scattered through a material or target sample when a monochromatic light beam strikes it. When exposing the sample to the beam of light, the molecules of this acquire energy and move to higher energy levels, producing vibrational and rotational movements that depend on the composition of the molecules and the strength of bonds between them, producing a characteristic spectral image [14]. In the spectrum produced, each band corresponds to a component of the sample; the position, intensity, displacement, and change in the shape of the bands provide structural information about the sample [15]. A previous study evaluated in vitro cervical tissue Raman spectra in different conditions. They reported that the overall accuracy of Raman spectroscopy for determining if a patient had cervical disease was higher than 99%. They also found subtle spectral differences between normal cervical epithelium and mild HPV changes (low-grade lesions), mainly in the region of 1334 cm\({}^{-1}\) and 1082 cm\({}^{-1}\), bands associated with the content of cellular DNA [15]. Other studies have described spectral differences using HPV positive and HPV negative cell lines. HPV negative (C33A) cells and HPV-positive (HeLa and SiHa) cells showed differences at amide I and amide III region, reporting a Raman spectroscopy classification efficiency of -95 [16; 17]. Many of the studies reported in the literature have focused on cell pellets instead of individual cells. There are also studies that explored Raman spectroscopic approach to discriminate malignant exfoliated cervical cell pellets. There has been reported a classification efficiency of about 79% and 78% for normal and abnormal smear, respectively, comparable to Pap test [18]; in the spectrum of normal cervical cells, bands corresponding to amide I, CH\({}_{2}\) stretches, and ring proteins such as phenylalanine (1660, 1450 and 1002 cm\({}^{-1}\)) predominate; on the contrary, in the spectrum of abnormal cells, a predominance of heme and fibrin bands is observed (1620 and 1570 cm\({}^{-1}\)) [18]. Some studies have examined individual cells and explored the biochemical patterns in cervical cells' nucleus and cytoplasm. Raman spectroscopy applied to exfoliated cervical cells may discriminate specific biochemical patterns identifying cell types, premalignant stages, and cancer [16; 18; 19]. Other studies have evaluated the bands corresponding to specific cellular components, such as the one carried out in 2020, in which the decrease in the intensity of bands corresponding to glycogen in cervical cells infected with HPV was observed, which was associated with an increase in energy needs for viral protein synthesis and replication [20]. In a recent study (2021), spectral differences were obtained that allowed discriminating between samples with active and non-active HPV infection, which was based predominantly on changes such as the increase in the intensity of nucleic acid bands (1485 and 1580 cm\({}^{-1}\)) and decreased glycogen bands (1334 and 1380 cm\({}^{-1}\)), changes caused by overexpression of viral proteins in cells with active viral infection [21]. In a study by [PERSON] et al. (2021), spectral differences between cervical tissue of different grades of CIN, squamous cell carcinoma, and adenocarcinoma were elucidated, observing Raman bands corresponding to lipids, amino acids, collagen, and cytosine of greater intensity depending on the degree of inflammation of the tissue, being greater in high-grade lesions and cancer [22]. Another study compared spectral characteristics between cervical tissue infected with HPV and tissue from invasive carcinoma using SERS, with gold nanoparticles as substrate (Supplemental Material) [23]. Spectral differences were reported in the region 920-1140 cm\({}^{-1}\) (lower intensity in carcinogenic vs. HPV).