## 3 Results and Discussion  
### Sub-Diffraction Raman Imaging on Single Cells of E. coli  
In order to determine the spatial distribution of biocomponents of an _E. coli_ ATCC 8739 cell Raman imaging was performed on a randomly selected isolated bacterial cell found in a drop of _E. coli_ with a concentration of \(1\times 10^{8}\) Colony-forming Units(CFU)/mL deposited on a golden substrate. Firstly, the mean Raman spectrum of _E. coli_ was acquired (Figure 1) to present the complete chemical fingerprint of the bacterium. A tentative assignment of the main Raman bands to the molecular functional groups and the corresponding biomolecules is reported in Table 1.  
Figure 1: Raman spectra of a single cell of _E. coli_ spotted on a golden substrate with respective band assignment. Then, an air-dried (Figure 2A(\(\alpha\))) and a freeze-dried (Figure 2A(\(\varphi\))) _E. coli_ sample were compared to determine the different distribution of biological components, as captured by sub-micrometrical Raman imaging, in differently treated cells. This provides the possibility to perform an intracellular investigation of the spatial distribution of biological components using a hyperspectral technique without staining. The image of the single _E. coli_ cell was obtained in a scanning area of about 6 \(\times\) 5 \(\upmu\)m. For both samples, the collection conditions were optimized, maximizing the most intense Raman signal at 2940 cm\({}^{-1}\). The maps were colored according to the intensity of the peak of interest from blue to red as the Raman signal increased, to easily localize the corresponding biomolecules within the cell area. In Figure 2B, Raman images colored with respect to the intensity of the most intense band in the Raman spectra of bacteria at 2940 cm\({}^{-1}\) are shown, this signal corresponds to CH\({}_{2}\)/CH\({}_{3}\) stretching [20; 21]. These images attest that the cell is mostly intact after drying and that the biomaterial is uniformly distributed into the cell both in the air-dried and in the freeze-dried cells, even though a higher intensity can be noticed for the air-dried cell. Greater differences between the air-dried and the freeze-dried cells were revealed when focusing the attention on the peak at 1449 cm\({}^{-1}\) (Figure 2C) that is related to \(\delta\)(CH\({}^{2}\)/CH\({}_{3}\)) modes of C-H functional groups present in lipids, amino acid side chains of the proteins, and carbohydrates, which are all mostly located on the bacterial membrane region [24]. Differences in the integrity of the membrane were expected since bacteria subjected to freeze-dry process were instantly frozen in liquid nitrogen without the addition of any cryoprotective agent in this study, while air-dried bacteria did not experience any thermal shock and preserve a more intact membrane. In Figure 2A(\(\varphi\)), we show a freezed cell, which looks optically intact. Nevertheless, the distribution of the biocomponents revealed by Raman imaging (Figure 2B-E(\(\varphi\))) witnesses membrane damage due to the sudden freeze shock. Even though freeze-drying method in the presence of a cryoprotectant could lead to a highly preserved cell structure, as it will be probably explored in a future research work, this first experiment was useful to demonstrate that Raman images capture the real distribution of molecular species within the sample, revealing differences in the case of proper or poor preparation method. It can be noticed that in the air-dried sample