# Raman Spectroscopy of Individual Cervical Exfoliated Cells in Premalignant and Malignant Lesions
## 3 Results  
Representative Raman spectra of the cytoplasm and cervical cell nuclei obtained from cervical exfoliation samples from healthy and CIN patients are shown in Figure 1A,B. The intensity of the Raman scattering in the cytoplasm and nucleus was increased in CIN patients compared to healthy patients. The main differences were in bands corresponding to proteins and nucleic acids. In the Raman spectra obtained from the cytoplasm of CIN patients compared to healthy patients, several characteristic bands stood out: 1077 cm\({}^{-1}\) (symmetric PO\({}_{2}\)\({}^{-}\) stretching of DNA), 1105 cm\({}^{-1}\) (cytosine), 1320 cm\({}^{-1}\) (guanine (RNA/DNA), -CH groups in protein and carbohydrates), 1440 cm\({}^{-1}\) (CH\({}_{2}\) proteins and lipid bending), 1546 cm\({}^{-1}\) (amide II), 1600 cm\({}^{-1}\) (adenine, guanine), and 1603 cm\({}^{-1}\) (C=C phenylalanine and tyrosine bond). The Raman spectra were normalized using the highest band of the entire set of samples as the reference value, corresponding to a high-grade lesion (Raman shift of 1440 cm\({}^{-1}\), in cytoplasm and nucleus). The nucleic acid bands of CIN patients also present increased intensities at 861 cm\({}^{-1}\) (phosphate group), 1320 cm\({}^{-1}\) (RNA/DNA guanine), and 1603 cm\({}^{-1}\) (adenine and guanine). In addition, some bands corresponded to Raman assignments of lipids and carbohydrates (1396 and 1440 cm\({}^{-1}\)), and another band at 1546 cm\({}^{-1}\) and 1650 cm\({}^{-1}\) corresponded to Amide II and Amide I, collagen, and protein, respectively [13; 28; 29; 30]. Conversely, when analyzing the spectra corresponding to the nucleus, the bands in which their intensity increased were similar to those of the cytoplasmic spectra. Still, some frequently appear red-shifted and correspond to proteins and lipids. Raman frequencies, as well as the molecular characteristics assigned to them, are shown in Table 1.  
Figure 1: Raman spectra of exfoliated cervical cells from healthy and CIN patients. (**A**) Average spectrum obtained from the cervical cell cytoplasm of healthy patients (blue line) and that of patients with a cervical lesion, CIN (red line). (**B**) Average of Raman spectra of the cervical cell nucleus of a healthy patient (blue line) and a patient with lesion, CIN (red line). The most prominent bands are shown for both cytoplasm and nucleus spectra. The SIMPER analysis yielded the main bands contributing to 50% of the statistical significance. Some can be seen to increase in intensity in Figure 1A. These bands were: 1077, 1108, 1320, 1440, 1546, 1603, and 1608 cm\({}^{-1}\). Table 1 summarizes the Raman assignment of all observed bands, whether in the cytoplasm or the nucleus. In addition, the differences in the spectra obtained from the cell nucleus were significant (Pseudo F = 4.60, \(p\) = 0.0039). The bands contributing 50% of the significant difference were the following: 861, 1323, 1394, 1440, 1574, 1600, and 1648 cm\({}^{-1}\) (Figure 1B). Figure 2 shows two graphs corresponding to MDS and bootstrap averages of the Raman cytoplasm and nucleus spectra of the two groups of patients studied: healthy patients (blue) and patients with a cervical lesion or CIN (red).