*3.1. Discrimination of HF Structures, Distribution of HSPG and GPC1 Using IR Spectral Imaging Analysis*

Anagen, catagen, and telogen HFs were individually isolated and maintained in culture for one day (A1, C1, and T1, respectively) or three days (A3, C3, and T3, respectively) before analysis. The white light image in Figure 2A shows a section of a HF and its different structures: germinative matrix (1), differentiation zone (2), IRS (3), ORS (4), and hair shaft (5). OCT is indicated by number 6.

**Figure 2.** PCA analysis of spectra from specific hair follicle regions. (**A**) White light image of a hair follicle. Five hair follicle structures were analyzed: 1, germinative matrix; 2, differentiation zone of the matrix; 3, IRS; 4, ORS; 5, hair shaft. The number 6 corresponds to OCT. (**B**) Pixel spectrum representative of each structure and OCT. (**C**,**D**) PCA score plot performed on normalized mean spectra of the above regions in the 1800–900 cm−<sup>1</sup> (**C**) or 1350–1190 cm−<sup>1</sup> (**D**) range and carried out on anagen A1/A3, catagen C1/C3, and telogen T1/T3 hair follicles. Ellipses represent the 95% confidence intervals.

3.1.1. Different Structures of the Hair Follicle are Discriminated by IR Spectral Imaging Analysis Based on the Variation of GAGs and Their Sulfation

The whole section was analyzed by IRSI and representative spectra of the HF structures are illustrated in Figure 2B. These spectra represent the means calculated from a small zone from each HF structure (indicated by 1 to 5). For comparison, the mean spectrum of OCT was also represented (6). The analysis of the GAG spectral profiles in the different HF structures was performed by PCA first in the 1800–900 cm−<sup>1</sup> spectral range (Figure 2C) corresponding to the proteins and PGs and then in the 1350–1190 cm−<sup>1</sup> spectral range (Figure 2D), focusing on sulfated GAGs. Figure 2C shows the PCA score plot built with the principal components PC1 and PC3 carrying 58.4% and 13.3% of the total explained variance, respectively. This score plot revealed five structural groups distributed in three well-differentiated zones. The first zone was composed of an overlap of the germinative matrix (group 1) and the differentiation zone (group 2). This overlap can be explained by the fact that the matrix cells of the differentiation zone are the result of differentiation of the progenitor cells present in the germinative matrix derived from stem cells of SHG [36–39]. They therefore share similar characteristics, such as proteins and polysaccharides, explaining their similarity. The second zone is constituted by the ORS (group 4), the third—by the hair shaft zones of HFs (group 5). It can be noted that the IRS structure (group 3) lies between the ORS and the hair shaft zones. Interestingly, IRS histologically separates the hair shaft from the ORS, which corroborates spectral data.

Figure 2D shows the PCA score plot built with the principal components PC1 and PC2 carrying 94.4% and 3.9% of the total explained variance, respectively. The PCA performed in the sulfated GAG absorption range (1350–1190 cm−<sup>1</sup> ) succeeded to precisely discriminate the germinative matrix structure (group 1) from the four other groups and, in particular, from the differentiation zone (group 2), in contrast with the overlap observed in Figure 2C. The other structures (differentiation zone, IRS, ORS, and hair shaft) were not clearly separated using sulfated GAG spectral signatures. Interestingly, different groups obtained by this PCA reflect the scheme of differentiation of the multipotent HF cells [36–39]. Indeed, the progenitor hair cells present in the germinative matrix provide, on the one hand, the ORS cells and, on the other hand, the transit-amplifying cells of the differentiation zone that will differentiate into IRS and hair shaft cells.

The PCA results shown in Figure 2C,D allowed discriminating several features of the HFs, the germinative matrix, the differentiation zone, the ORS, the IRS, and the hair shaft, by the contribution of proteins, PGs, GAGs, and sulfated GAGs. The biochemical spectral information is confirmed by the loading vectors shown in the region of 1800–900 cm−<sup>1</sup> (Figure S1A) and in the region of 1350–1190 cm−<sup>1</sup> (Figure S1B). The sulfate group vibrations of GAGs are mainly represented by the IR absorption band centered at 1248 cm−<sup>1</sup> . This peak is assigned an antisymmetric stretching S=O vibrations as we have previously reported [29]. This discrimination is in agreement with the difference in PG composition in the different parts of HFs observed by immunostaining or immunofluorescence [21,22,40]. Moreover, the IRSI technique does not require any staining or chemicals while keeping good discrimination of histological structures.

3.1.2. Focus on GPC1 Distribution in Hair Follicles by Spectral Imaging Analysis and Immunohistochemistry

In order to better understand the contribution of HSPGs in the differences observed by the PCA in the spectral range corresponding to PGs/GAGs and sulfated GAGs, a correlation image was computed with a spectrum of standard HS. This analysis permitted obtaining a representative image of the HS contribution in the section of HFs. As shown in Figure 3, the coefficient of HS contribution was most important in the ORS. The IRS displayed a good correlation with HS. The germinative matrix structure presented a low correlation with HS, while the differentiation zone and the hair shaft did not correlate with the HS. This result correlates well with HS distribution in the HFs obtained by immunohistochemistry reported by Malgouries et al., where several HSPGs were found in the ORS part of HFs [21,40].

**Figure 3.** Characterization of hair follicle structures by different imaging approaches. From left to right: white light image, HS- and GPC1-correlated IR images, and immunohistochemical labeling of GPC1 (green) counterstained with the Evans blue dye (red). CTS, connective tissue sheath; IRS, inner root sheath; ORS, outer root sheath. Arrowheads indicate high level of correlation. Scale bar: 100 µm.

A correlation image was also created with a representative spectrum of GPC1. The coefficient of GPC1 contribution is most important in the differentiation zone (Figure 3). The hair shaft, the ORS, and the germinative matrix also presented a good correlation with GPC1, but not the IRS. The GPC1 correlation map is confirmed by the GPC1 green labeling obtained by immunohistochemistry of HFs. These results obtained by the IRSI technique demonstrate its ability to discriminate at the molecular scale in a comparable manner to immunohistochemistry without any labeling.

These results support the PCA analyses carried out on the different HF structures. The use of both HS and GPC1 correlation mapping may partly explain the discrimination potential of the PCA analyses.
