Confocal Raman imaging of skin containing hair follicles

Confocal Raman imaging of skin containing hair follicles J. Schleusener, V. Carrer, A. Patzelt, S. Guo, T. Bocklitz, J. Lademann, M. E. Darvin UNIVERSITÄTSMEDIZIN BERLIN maxim. darvin@charite. de 1

Overview Department of Dermatology, Venerology and Allergology, Charité – Universitätsmedizin Berlin www. ccp-berlin. org § Introduction Confocal-Raman Microscopy (CRM) § Experimental procedure § Sample surface determination § Distribution of compounds § Results § Conclusion UNIVERSITÄTSMEDIZIN BERLIN 2

Confocal Raman Microscopy (CRM) Introduction § Irradiation of a sample with light, inelastic scattering is observed § Energy transfer can be measured as shift of wavelength § This Raman shift is molecular specific (functional groups) Main Features § Molecular specificity § non-invasive § in vivo or ex vivo § No sample preparation required § high spatial resolution to analyze different layers of the skin § Imaging can be achieved by scanning sample (long acquisition times) UNIVERSITÄTSMEDIZIN BERLIN 3

Confocal Raman Microscope (CRM) Horiba Jobin Yvon, Lab. RAM HR Evolution (Longjumeau Cedex, France) § Excitation at: § 325 nm (8. 1 m. W) § 473 nm (6. 1 m. W) § 633 nm (8. 4 m. W) § Detection range adjustable (FP + HWN) § Spectral resolution: <4 cm-1 § Only applicable ex vivo Mujica Ascencio et al. Eur. J. Pharm. Biopharm. 104: 51 -58, 2016 UNIVERSITÄTSMEDIZIN BERLIN 4

Hair follicles Microscopic imaging of hair follicles § Contain a multitude of different skin and hair components § § § Dermis Viable Epidermis Stratum corneum (SC) Hair Sebum § Are of interest for the research of different penetration routes for topically applied substances § Intercellular § Intracellular § Follicular UNIVERSITÄTSMEDIZIN BERLIN 5

Experimental procedure § Porcine skin (model substance for human skin) was obtained from local butcher § Bristles were carefully removed without influence on the SC § Biopsies were taken and fixed with cryospray and liquid nitrogen and subsequently frozen at -20 °C § 300 µm thick cryosections with hair follicles were prepared § Mapping with the Confocal-Raman microscope § Data analysis § Sample surface determination (autofocus) (based on reflection or spectral distribution) § Imaging of component distribution (MCR-ALS and CLS) UNIVERSITÄTSMEDIZIN BERLIN 6

Analysis with CRM Acquisition conditions § Excitation at: 473 nm, 6. 1 m. W § Detection in the Finger Print region (400 -2000 cm-1) keratin § Multitude of characteristic Raman bands § Spectral resolution: <4 cm-1 § Only applicable ex vivo § 5 sec/spectrum, 100 x objective lipids (depth 38 µm) § Imaging of hair follicle Finger print region UNIVERSITÄTSMEDIZIN BERLIN High Wavenumber region 7

Surface determination: reflection § Surface is determined based on intensity of reflected signal § Spectra are only obtained from surface position of Cryosections Epidermis UNIVERSITÄTSMEDIZIN BERLIN Depth Dermis Hair 8

Surface determination: spectral distribution AUC (count) § Multiple spectra are recorded at different z-positions § Surface is determined based keratin profile Depth (μm) Schleusener et al. Laser Physics Letters 14: 125601, 2017 UNIVERSITÄTSMEDIZIN BERLIN 9

Molecular Compounds in Cryosections Glass Hair SC Epidermis Dermis Cryospray § Spectra of reference substances were applied as initial loadings for further analysis § Analysis of single band intensities is not useful due to overlapping of different components UNIVERSITÄTSMEDIZIN BERLIN 10

Distribution of Compounds Classical least-squares (CLS) § § Labspec software Number of sample components known The reference spectra of the components are acquired Resulting Scores: % contribution of the reference spectra Glass Hair SC Epidermis Dermis Cryospray UNIVERSITÄTSMEDIZIN BERLIN 11

Distribution of Compounds Multivariate Curve Resolution. Alternative Least-Squares (MCR-ALS) § Iterative optimization § No need of reference spectra § Can take reference spectra as Dermis Epidermis SC Hair starting values § Sensitive to peak shifts Glass Cryospray UNIVERSITÄTSMEDIZIN BERLIN 12

Conclusions: § Skin sections containing hair follicles have been analyzed by CRM. § The analysis was based on multivariate data analysis for detection of the spatial distribution of the components. § Confocal Raman microscopy combined with the CLS or the MCR-ALS data analysis enables visualization and characterization of the most important components of skin sections containing hair follicles without any labeling or staining. Future research: § Application of presented method for analysis of penetration routes of topically applied substances. § For validation, cryosections with known distribution of penetrated substances are necessary. UNIVERSITÄTSMEDIZIN BERLIN 13

Thank you for your attention UNIVERSITÄTSMEDIZIN BERLIN 14