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Characterizing Heterogeneous Cellular Responses to Polymethoxyflavones Using Raman Microscopy

To understand how individual cells respond to perturbations is a challenge as much of our knowledge is based on ensemble measurements. While cellular heterogeneity cannot be ignored, a practical analytical tool for analyzing and interpreting cellular heterogeneity is needed. Herein, we report the use of Raman Spectroscopy to characterize heterogeneous cellular responses of human colon cancer cells to polymethoxyflavones (PMFs), a group of anti‐cancer flavonoids found in citrus fruits. Raman spectroscopy is a molecular vibrational spectroscopy can measure chemical signatures of a single cell noninvasively. In this study, the cells were treated with PMFs, i.e. nobiletin (NBT) and 5‐demethylnobiletin (5DN) for 24, 48 and 72 hours, and both the floating and the attached cells were collected for Raman measurement. Heterogeneity of their Raman spectra was analyzed by principal component analysis (PCA). The results showed that Raman Spectroscopy could clearly characterize the difference between floating and attached cells under different treatments and at different treatment time. The biochemical profiles of floating cells are more heterogeneous in protein compared with those of the attached cells, indicating the different sensitivity and behavior of individual cells responding to NBT or 5DN. This phenomenon was less pronounced when cells were treated for longer period of time (72 hours vs. 48 hours). Our findings demonstrated that Raman microscopy offers a reliable method for characterizing individual cells, which can be used to investigate the heterogeneous responses of cell population to environmental stimuli.