**4. Conclusions**

This report demonstrates the capacity of IRSI to identify the different tissue structures of the HFs and to show protein, PG, GAG, and sulfated GAG distribution in these structures. In addition, the comparison between the anagen, catagen, telogen phases of the hair growth cycle shows the qualitative and/or quantitative evolution of GAGs as supported by Western

immunoblotting. Thus, IRSI can reveal some information on the location of proteins, PGs, GAGs and sulfated GAGs in HFs in a reagent- and label-free manner. In the long term, from a dermatological point of view, IRSI might constitute a promising technique for the early diagnosis and prevention in alopecia.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/2218-273 X/11/2/192/s1, Figure S1: PCA analysis of spectra from specific hair follicle regions. (A-B) Loading vectors 1 and 3 obtained from Figure 2C (A) and loading vectors 1 and 2 obtained from Figure 2D (B), Figure S2: Contribution of hyaluronic acid in hair follicle. (A) Spectrum of standard HA. (B–D) IR correlation maps of hair follicles in anagen A1 (B), catagen C1 (C) and telogen T1 (D) phases using spectrum from standard hyaluronan. HA, hyaluronan. Arrowheads indicate good level of correlation. Scale bar: 100 µm, Figure S3: PCA analysis of spectra from specific hair follicle regions. (A–C) Loading vectors 2 and 3 obtained from Figure 6A (A), loading vectors 1 and 2 obtained from Figure 6B (B), and loading vectors 1 and 2 obtained from Figure 7A (C).

**Author Contributions:** Conceptualization, S.B., L.R., and G.D.S.; methodology, C.C-P., V.U., S.B., L.R., and G.D.S.; software, V.U.; validation, S.B., L.R., G.D.S., N.B., L.D., V.B., S.M. and C.J.; formal Analysis, C.C.-P., V.U., S.B., L.R., and G.D.S.; investigation, C.C.-P., V.U., S.B., L.R., and G.D.S.; resources, N.B.; data curation, C.C.-P., V.U., S.B., L.R., and G.D.S.; writing—original draft preparation, C.C.-P., V.U., S.B., L.R., and G.D.S.; writing—review and editing, C.C-P., V.U., S.B., L.R., G.D.S., N.B., L.D., V.B., S.M. and C.J.; supervision, S.B., L.R., and G.D.S.; project Administration, S.B., L.R., and G.D.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was made in collaboration with BASF Beauty Care Solutions. Ms Charlie Colin–Pierre is a BASF/CNRS (Centre national de la recherche scientifique)-funded PhD fellow.

**Institutional Review Board Statement:** In our study, commercial human scalp samples were ordered and provided by Alphenyx (Marseille, France) after surgery of human donors following informed consent, and applicable ethical guidelines and regulations.

**Informed Consent Statement:** Informed consent was obtained by Alphenyx from all subjects involved in the study.

**Data Availability Statement:** Not Applicable.

**Acknowledgments:** The authors thank the PICT-IBiSA platform of the University of Reims Champagne–Ardenne for instrument facilities. We wish to thank Carine Tedeschi for her technical support in performing hair follicle sections and immunohistochemistry and Nathalie Andres for isolation and culture of hair follicles.

**Conflicts of Interest:** The authors declare no competing financial interest.
