## 3 Results and Discussion
### Sub-Diffraction Raman Imaging on Single Cells of E. coli
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Raman Shift (cm\({}^{-1}\))** & **Assignment 1** & **Chemical Compound 1** & **Reference** \\ \hline
2940 & \(\
u\)CH\({}_{2}\) asymmetric & carbohydrates, amino acids, proteins, & 2935 [20] \\  & \(\
u\)CH\({}_{3}\) symmetric & lipids & 2935 [21] \\ \hline
1662 & amide I band & nucleic acids & 1666 [22] \\  & \(\
u\)C=C & & 1663 [21] \\ \hline
1573 & amide II band & nucleic acids (G, A) & 1565 [23] \\  & \(\delta\)NH; \(\
u\)CN & 1575 [20] \\ \hline
1440-1480 & \(\delta\)(CH\({}_{2}\)/CH\({}_{3}\)) & lipids, amino acid, side chains of proteins and carbohydrates & 1440–1460 [20]; 1450 [22] \\ \hline
1332 & ring vibration & nucleic acids (G, A) and tryptophan & 1337 [20] \\  & \(\delta\)CH\({}_{2}\) & proteins & 1337 [20] \\ \hline
1243 & \(\
u\)C–C, \(\
u\)C–O amide III band & nucleic acids (A, T) & 1250–1000 [23] \\  & \(\
u\)(PO\({}_{2}\)\({}^{-}\)) asymmetric & & 1245 [20] \\  & \(\
u\)C–O-S & \(\
u\)Nolipids & 1243 [20] \\ \hline
1098 & ring stretching & saccharide components (lipid, DNA and RNA backbone); phenylalanine & 1115–1005 [23] \\  & \(\
u\)C–O–C symmetric glycosidic & & 1104 [21] \\  & link; \(\
u\)C–N; \(\
u\)C–C & & 1125 [20]; 1099 [21] \\ \hline
780 & ring breathing modes & nucleic acids (C, T, U), phenylalanine, & \\  & & pyranose compounds & 786–780 [21] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Raman bands assignments to functional groups and corresponding biomolecules. (Figure 2C(\(\alpha\))), the highest Raman intensity areas (red to yellow) are more homogeneously spread throughout the whole bacterial cell than in the freeze-dried sample (Figure 2C(\(\varphi\))), where the membrane seems to have been damaged and the shape of the bacterial membrane results less defined. The line profiles of intensity across the cell (both longitudinal and transversal) for the same signals were extracted and normalized. They are plotted together in Figure S1 in supplementary information (SI) for an alternative visualization of the differences in the spatial distribution of the considered molecules. Complementary analysis was done, recoloring the Raman images based on the signal intensity at 1243 cm\({}^{-1}\) (Figure 2D) and 780 cm\({}^{-1}\) (Figure 2E), which refer specifically to nucleic acids vibrations [21; 23; 24], a similar statement would be probably possible considering also the vibrational modes around 1000-1100 cm\({}^{-1}\), but its less specific assignation and  
Figure 2: (**A**) Confocal Microscope image of a sample of _E. coli_ spotted on a gold surface taken with a 100\(\times\) microscope objective. (**B**) Chemical Raman map obtained on the single bacterial cell; the color scale is based on the intensity of the Raman signal at 2940 cm\({}^{-1}\) of generic biomaterial. (**C**) Chemical Raman map at 1449 cm\({}^{-1}\) representing cell membrane. (**D**) Chemical Raman map at 1243 cm\({}^{-1}\) representing nucleic acids. (**E**) Chemical Raman map at 780 cm\({}^{-1}\) representing nucleic acids; (**\(\alpha\)**) air-dried cell, (**\(\varphi\)**) freeze-dried cell. the possible overlapping with saccharide components led us to conveniently consider the 780 cm\({}^{-1}\) peak for DNA mapping. As it is shown in Figure 2D and 2E, the spatial separation of the regions of the _E. coli_ cell, in which the signal related to DNA vibrational modes shows relatively high intensity, is different from those characterized by the higher C-H signals of the membrane. Also in this case, it is clear that the air-dried sample (Figure 2D(\(\alpha\)),E(\(\alpha\))) presents a more homogeneous distribution of the high-intensity regions than the freeze-dried sample (Figure 2D(\(\varphi\)),E(\(\varphi\)), respectively). Moreover, while in the air-dried cell the signal is condensed in the center of the cell, which is in accordance with the biological distribution of genetic material, in the freeze-dried sample it is not well confined by an intact membrane but it is inhomogeneously spread around, as it happens after membrane disruption.