Phenotype Identification of HeLa Cells Knockout CDK6 Gene Based on Label-Free Raman Imaging

Identifying cell phenotypes is essential for understanding the function of biological macromolecules and molecular biology. We developed a noninvasive, label-free, single-cell Raman imaging analysis platform to distinguish between the cell phenotypes of the HeLa cell wild type (WT) and cyclin-dependent kinase 6 (CDK6) gene knockout (KO) type. Via large-scale Raman spectral and imaging analysis, two phenotypes of the HeLa cells were distinguished by their intrinsic biochemical profiles. A significant difference was found between the two cell lines: large lipid droplets formed in the knockout HeLa cells but were not observed in the WT cells, which was confirmed by Oil Red O staining. The band ratio of the Raman spectrum of saturated/unsaturated fatty acids was identified as the Raman spectral marker for HeLa cell WT or gene knockout type differentiation. The interaction between organelles involved in lipid metabolism was revealed by Raman imaging and Lorentz fitting, where the distribution intensity of the mitochondria and the endoplasmic reticulum membrane decreased. At the same time, lysosomes increased after the CDK6 gene knockout. The parameters obtained from Raman spectroscopy are based on hierarchical cluster analysis and one-way ANOVA, enabling highly accurate cell classification.