Carvalho Luis Felipe C S, Bonnier Franck, Tellez Cláudio, Dos Santos Laurita, O'Callaghan Kate, O'Sullivan Jeff, Soares Luis Eduardo S, Flint Stephen, Martin Airton A, Lyng Fiona M, Byrne Hugh J
FOCAS Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8, Ireland; Departamento de Odontologia da Universidade de Taubaté (UNITAU), Brazil.
Université François-Rabelais de Tours, Faculté de Pharmacie, EA 6295 Nanomédicaments et Nanosondes, 31 avenue Monge, 37200 Tours, France.
Exp Mol Pathol. 2017 Dec;103(3):255-262. doi: 10.1016/j.yexmp.2017.11.001. Epub 2017 Nov 7.
Raman spectroscopy can provide a molecular-level signature of the biochemical composition and structure of cells with submicrometer spatial resolution and could be useful to monitor changes in composition for early stage and non-invasive cancer diagnosis, both ex-vivo and in vivo. In particular, the fingerprint spectral region (400-1800cm) has been shown to be very promising for optical biopsy purposes. However, limitations for discrimination of dysplastic and inflammatory processes based on the fingerprint region have been demonstrated. In addition, the Raman spectral signal of dysplastic cells is one important source of misdiagnosis of normal versus pathological tissues. The high wavenumber region (2800-3600cm) provides more specific information based on NH, OH and CH vibrations and can be used to identify the subtle changes which could be important for discrimination of samples. In this study, we demonstrate the potential of the high-wavenumber spectral region in this context by collecting Raman spectra of nucleolus, nucleus and cytoplasm from oral epithelial cancer (SCC-4) and dysplastic (DOK) cell lines and from normal oral epithelial primary cells, in vitro, in water immersion, which were then analyzed by principal components analysis as a method to discriminate the spectra. Analysis was performed before and after digital subtraction of the bulk water signal. In the normal cell line, the three subcellular regions are well differentiated before water subtraction, although the discrimination of the two nuclear regions is less well defined after water subtraction. Comparing the respective subcellular regions of the three cell lines, before water subtraction, the cell lines can be discriminated using sequential PCA and Feature Discriminant Analysis with up to ~100% sensitivity and 97% specificity for the cytoplasm, which is improved to 100% sensitivity and 99% specificity for the nucleus. The results are discussed in terms of discrimination comparing the CH vibrational modes of nucleic acids, proteins and lipids. The potential role of the OH vibrations, considering free water and confined water, in the discrimination of cell cultures and pathological processes are also discussed.
拉曼光谱能够以亚微米级的空间分辨率提供细胞生化组成和结构的分子水平特征，对于监测成分变化以进行早期非侵入性癌症诊断（包括离体和体内诊断）可能很有用。特别是，指纹光谱区域（400 - 1800cm）已被证明在光学活检方面非常有前景。然而，基于指纹区域区分发育异常和炎症过程存在局限性。此外，发育异常细胞的拉曼光谱信号是正常组织与病理组织误诊的一个重要来源。高波数区域（2800 - 3600cm）基于NH、OH和CH振动提供更具体的信息，可用于识别对样本区分可能重要的细微变化。在本研究中，我们通过在体外水浸条件下收集口腔上皮癌（SCC - 4）、发育异常（DOK）细胞系以及正常口腔上皮原代细胞的核仁、细胞核和细胞质的拉曼光谱，展示了高波数光谱区域在这方面的潜力，然后通过主成分分析作为区分光谱的方法进行分析。在对大量水信号进行数字扣除前后均进行了分析。在正常细胞系中，在扣除水之前，三个亚细胞区域区分良好，尽管扣除水后两个核区域的区分不太明确。比较三个细胞系各自的亚细胞区域，在扣除水之前，使用顺序主成分分析和特征判别分析可以区分细胞系，对于细胞质，灵敏度高达约100%，特异性为97%，对于细胞核，灵敏度提高到100%，特异性为99%。根据核酸、蛋白质和脂质的CH振动模式比较区分结果进行了讨论。还讨论了考虑自由水和受限水的OH振动在细胞培养物区分和病理过程中的潜在作用。
