## 3 Results and Discussion
### Analysis of SERS Spectra  
SERS spectral changes between the control group and the cordycepin-12 h group are shown in Figure 3. Trace (a) represents the average SERS spectra of the control group and trace (b) is the average SERS spectra of the cordycepin-12 h group. The corresponding spectral difference between the cordycepin-12 h group and the control group is shown as trace (c). The SERS spectral changes between these two groups represent content changes in the early stage of apoptosis. From Figure 3 trace (c), obvious differences between the cordycepin-12 h group and the control group at SERS characteristic peaks 534, 640, 746, 805, 830, 998, 1145, 1438, and 1546 cm\({}^{-1}\) can be clearly observed. The spectral peak assignments are listed in Table 1. Compared to control cells, SERS peak intensities in apoptotic cells showed increases at 534, 746, and 1145 cm\({}^{-1}\), which could be assigned to A ring (adenine ring) breath, T ring (thymine ring) breath, and deoxyribose phosphate, respectively. This indicated that nucleic acid concentration had increased in the early stage of cordycepin-induced apoptosis. On the other hand, peaks at 640, 805, 830, 998, 1438, and 1546 cm\({}^{-1}\) exhibited decreases in intensities in apoptotic cells  
Figure 2: Differential interference contrast (DIC) and fluorescence microscopic images of HeLa cells. The DIC images of cells from: (**a**) the control group, (**d**) cordycepin-12 h group, (**g**) cordycepin-48 h group; the fluorescence images of cells from: (**b**) the control group, (**e**) the cordycepin-12 h group, (**h**) the cordycepin-48 h group; the merged DIC and fluorescence images are shown in (**c**), (**f**), and (**i**), respectively. compared to control cells. A shoulder peak appeared at 830 cm\({}^{-1}\), which was related to the stretching vibration of O-P-O asymmetric bond, also decreased in the cordycepin-12 h group. The peak at 640 cm\({}^{-1}\) was assigned to the C-C twist of tyrosine, the peak at 998 cm\({}^{-1}\) could be assigned to the symmetric ring breathing mode of phenylalanine, and the peak at 1546 cm\({}^{-1}\) was assigned to Amide II. The intensity decreases at these characteristic peaks revealed that the contents of proteins and lipids had decreased in the early stage of cordycepin-induced apoptosis. At the same time, the peaks at 805 and 1438 cm\({}^{-1}\), which were assigned to RNA, also decreased, indicating that the RNA concentration began to decline in the early stage of apoptosis. Figure 4 shows SERS spectral changes between the control group and the cordycepin-48 h group. The averaged SERS spectra of the control group are shown in trace (a), the cordycepin-48 h group is shown in trace (b), and the spectral differences between them is in trace (c). This figure exhibits the content changes in the late stage of cordycepin-induced apoptosis. Spectral differences at characteristic peaks 534, 746, and 1145 cm\({}^{-1}\) revealed intensity decreases for nucleic acid while spectral changes at peaks 640, 830, 998, and 1546 cm\({}^{-1}\) revealed intensity increases for proteins and lipids due to cordycepin-induced apoptosis at 48 h. Meanwhile, it was observed that peaks at 805 and 1438 cm\({}^{-1}\), which were assigned to RNA, encountered significant decreases in spectral intensity in the late stage of apoptosis. Figure S6 in the Supplementary Materials shows the averaged spectra and the difference spectra of all three groups in one graph. \begin{table}
\begin{tabular}{c c} \hline
**Raman shift (cm\({}^{-1}\))** & **Assignment** \\ \hline
534 & Adenine ring breath \\
640 & C–C twist of tyrosine \\
746 & Thymine ring breath \\
805 & RNA \\
830 & O–P–O asymmetric stretch, tyrosine ring breath \\
998 & Phenylalanine symmetric ring breath \\
1145 & Deoxyribose phosphate \\
1438 & RNA \\
1546 & Amide II \\ \hline \end{tabular}
\end{table} 
Table 1: Raman peak frequencies and their assignments. Figure 3: SERS spectral changes of HeLa cells treated with cordycepin in the early stage of apoptosis.