## 3 Results and Discussion
### Multivariate Curve Resolution-Alternating Least Squares  
MCR-ALS is an iterative modelling method that provides the optimized estimates of pure deconvolved spectral shapes (i.e., the loadings which maintain a profile with a chemical meaning of the original spectrum) and score matrix (representing the concentrations of each pure component in one single image pixel) to resolve mixed spectra, conserving the chemical meaning of each component. Figure 5 shows the MCR pattern of the two components that were found to contain major information in the MCR analysis. It is easy to notice that the MCR1 (Figure 5A) and MCR2 (Figure 5B) patterns are in accordance with PC1-PC2 score plot of PCA, but allow further interpretation of biomolecules distribution. As for what we have observed from the score plots, also in this case the spectral regions evidenced by MCR components have a high correspondence to that emerged from univariate analysis, but with higher specificity, since a spectral pattern is considered instead of one single peak at a time. Indeed, a spectral band analysis was possible on the MCR loadings plot, which allows easier comprehension with respect to PCA loadings. The MCR components indeed maintain a profile, which can be chemically interpreted thanks to the non-negativity constraint (Figure 6). Two MCR components were considered for the biochemical description of the system. Even though MCR3 explains 11.9% of variance, it was not considered since it is not related to meaningful spectral bands but to the baseline trend due to fluorescence compensation performed by the acquisition software and it is not relevant for the discussion. Figure 5: (**A**) Score plot of MCR1. The color scale is based on MCR1 intensity, from lowest (**blue**) to highest (**red**). (**B**) Score plot of MCR2. The color scale is based on MCR2 intensity, from lowest (**blue**) to highest (**red**). The MCR1 component shows spectral features mainly found in the spectra at the two poles of the bacterial cell, while the contribution of MCR2 is mainly revealed in the central region of the cell. Furthermore, higher MCR2 scores seem to concentrate on the border of the cell, indicating the correlation of those signals to the bacterial membrane. Moreover, the high contribution of CH band at 2940 cm\({}^{-1}\) in MCR1 suggests that there is a higher density of biomaterial in general, at the two poles of the cell (as previously mentioned), suggesting that the cell is undergoing replication. Cell division must be coordinated with the faithful segregation of the newly duplicated genetic material to each daughter cell. Precedent studies of the dynamic localization of proteins and genetic materials, conducted with fluorescence microscopy techniques in _E. coli_ cells [27], have shown that in the earliest stage of segregation, after origin duplication at the cell center, sister origins move rapidly toward opposite cell poles and remain there, which establishes polarity. Subsequent replication and DNA condensation result in the bulk of the chromosomes moving towards their respective origins [28]. In brief, during DNA replication, newly duplicated daughter chromosomes separate from each other, forming two large masses known as nucleoids with a DNA-free zone in between. Then, a cytokinesis ring made of tubulin-like proteins (FtsZ), called Z-ring, assembles approximately in the center of the cell; this ring is fundamental for bacterial replication because it recruits in an ordered sequence a series of additional proteins that mediate division. The nucleoids appear to interfere with Z-ring assembly, perhaps by close contact with the inside surface of the cytoplasmic membrane, where the nucleation of FtsZ polymerization is believed to take place [29]. The polymerization of the Z-ring protein is then favored in the cell middle, where chromosome segregation has created a space between the DNA masses. Thus, dynamic protein and nucleic acid determinants work together to ensure that the cell finds its precise mid-point when it undergoes binary fission [30] (pp. 89-98).