Raman Spectroscopy and imaging to explore skin and hair. What is the Raman scattering effect?

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1 Diapositive 1 1 Raman Spectroscopy and imaging to explore skin and hair Sir C.V. Raman Nobel price in physics in µm Diapositive 2 What is the Raman scattering effect? Incident monochromatic beam ν = 1/λ Scattered light is polychromatic Échantillon opaque ν + ν vib ; ν ; ν - ν vib + ν vib ; ; - ν vib Spectrum of bands shifted compared to the excitation position, cm -1 Échantillon transparent anti-stokes 35 cm -1 O-H élong 17 cm -1 C=O élong Stokes 17 cm -1 C=O élong 35 cm -1 O-H élong ν vib 2 ν λ

scattering UV-visible absorption E Fluorescence emission non-radiative relaxation 3 Diapositive 4 How to

2 Diapositive 3 Molecule-photon energy exchange v 2 v 1 v E 1 hν dif = hν hν vib États virtuels hν dif = hν hν dif = hν + hν vib v 2 v 1 v absorption IR Raman Stokes scattering Rayleigh scattering Raman anti-stokes scattering UV-visible absorption E Fluorescence emission non-radiative relaxation 3 Diapositive 4 How to record a Raman spectrum? - sample, no need of preparation - distant analysis is possible More or less simple intruments 4

3 Diapositive 5 Raman spectroscopy is complementary to IR, both being structure-specific methods Advantages in Raman : - no sample preparation (non destructive analysis, in situ) - distant analysis and in vivo analysis is allowed (optical fiber) - better spatial resolution (mixtures) and spatial resol-n (microscopy) - low frequencies are analysable (inorganic substances) 5 Diapositive 6 Difficulties in Raman - Poor sensitivity: 1-1 M (RR : 1-4 M) - interference with emissions 28 protein solution ~5g/L 26 Intensity (a.u.) protein powder Low signal from low density samples like cells (progres of instrumentation goes on) Use of powerful lasers emitting in red and near-ir

4 Diapositive 7 Raman spectrum from tissue Composed of the bands of: proteins lipids water DNA lipid lipid+protein protein A. Whitley and F. Adar. Cytometry Part A (26) 9A: Diapositive 8 Raman spectrum of a protein a) Primary structure: aromatic AA content Phe Intensity (a.u.) Cys 59 Fermi doublet: 853/829 increases upon Tyr exposure 563 Tyr Phe Tyr Trp Quantitation of the band area in the spectrum normalised at ~145 cm -1

5 Diapositive 9 Raman spectrum of a protein (BSA) a) Secondary structure (conformations) Intensity (a.u.) g-g-g g-g-t t-g-t Bridges S-S (C-C-S-S-C-C) υ(c-c) (α) Peptide bonds: Amide III C-H 2 scissoring 1448 Peptide bonds: Amide I hélix α Random coil 1683 sheet β Diapositive 1 Quantitative structure description pea globulin 12 random random 1654: 24 % 1669: 15 % 1683: 2 % hélix α 1654 random 1669 sheet β 1683 Intensity (a.u.) alpha gliadin hélice α hélice α Trp : 32 % 1669: 1 % 1683: 15 % Wavenumber (cm -1 ) Wavenumber (cm -1 ) % of conformations estimated by deconvolution of the Amide I band (Ducel et al., 26).

microscope (immobilisation against a quartz window d.

6 Diapositive 11 Raman measurements of skin in vivo Application note : horiba.com Laser 633,785 or 83 nm, 5-1 mw; obj 2X LWD or 5x LWD; Resolution in Z (Si): 6 to 3 µm Depth max ~3 µm Inversed microscope (immobilisation against a quartz window d.~2 mm) LabRam INV, Horiba JY Otherwise: optical fiber with focus/collect head Ex: Skrebova N. et al., J. Biomed. Opt. 25: 1, Diapositive 12 Raman spectra of skin in vivo Application note : horiba.com ν(c-h) ν(o-h) 83 nm 785 nm 633 nm 12

Diapositive 13 Hydration profiles non-hydrated hydrated After superficial application of a hydrating agent (cosmetology) pmpc (2-methacryloyloxethylphosphorylcholine polymer) with hyaluronic acid. L.

7 Diapositive 13 Hydration profiles non-hydrated hydrated After superficial application of a hydrating agent (cosmetology) pmpc (2-methacryloyloxethylphosphorylcholine polymer) with hyaluronic acid. L. Chrit et al. Biopolymers, 26, 85: Diapositive 14 FT-Raman of lipids in human stratum corneum (HSC) Lawson et al., Spectrochimica Acta Part A 54 (1998) C HSC whole HSC no lipids A: Band separation (cm -1 ) ν(ch 2 ) s and ν(ch 2 ) as 2847 and 2931 cm -1 of HSC for the temperatures C B: Thermal transition diagram 14 FT-Raman spectra of HSC: temperature: 25-1 C. B

Diapositive 15 Carotenoids in human skin Hata TR et al. J Invest. Dermatol. (2) 115, 441 448 Exc.

8 Diapositive 15 Carotenoids in human skin Hata TR et al. J Invest. Dermatol. (2) 115, Exc. 488 nm: strong fluorescence: correction Lutein, zeaxanthin, lycopene and its Z- isomers, and - α, -β, -γ, and ξ-carotene conc. quantitated by HPLC, compared with the 1524 cm -1 Raman intensity (counts). Carotenoid concentration in the skin correlates with the presence or absence of skin cancer and precancerous lesions 15 Diapositive 16 Human tissue : pathologic or not? 16

Diapositive 17 Raman spectroscopy of human

9 Diapositive 17 Raman spectroscopy of human hair: effect of chemicals (1) or of aging (2) (1) Kuzuhara A., Biopolymers, Vol. 77, (25) (2) Kuzuhara et al., Biopolymers Volume 87, (27) Diapositive Molecular imaging by Raman spectral mapping

8 7 6 5 4 3 2 1 1 µm 15 1 5 1 12 14 16 Diapositive 19 Principle of spectral

19 Contribution Raman, % naturels c12 à1% Statistics >1 maps Co-localisation with

correction mapping of intensity / form 2) Statistics Diapositive 2 Spatial

10 µm Diapositive 19 Principle of spectral mapping Spectra acquisition : ~5-1 sec per spectrum in Raman Sample video caption 19 Contribution Raman, % naturels c12 à1% Statistics >1 maps Co-localisation with TV image Parametric maps overlay Intensity (a.u.) Mean=37.4 Mean=62.6 Error= multi-spectral map ~9-25 spectra treatments : 1) Spectrals : baseline correction mapping of intensity / form 2) Statistics Diapositive 2 Spatial resolution of a confocal Raman microspectrometer the Raman intensity follows the cuticle layer Spectra from different layers -Cuticle -Cortex 1. -Medulla

11 Diapositive 21 Hair X-Y mapping algorithms : decomposition into spectral models µm Region-specific spectral models for decomposition Diapositive 22 Profiles of the hair composition and structure: fitting with gaussian profiles 2

12 Diapositive 23 Coloured gray hair Intensity (a.u.) Raman contribution, dye/protein, % Length Y (µm) Length X (µm) depth, µm Dye penetration profiles 23 Diapositive 24 Raman maps by spectra deconvolution Tissue after the sub-cutaneous implant of alginates in rat Mean=52.1 Mean=41.8 lipidm= 6.1 Error= 3.2 Spectra fitted with 3 models «DNA» (cells), «protein»(serum), «lipid» Contribution Raman, % µm naturels c12 à1% 24

13 Diapositive 25 Parametric maps of the same sample, corresponding to different Raman bands Models may be multiple, including inflammation, pathology, etc. 25 Diapositive 26 Acknowledgments Dr. Simone Cohen-Jonathan, Pr. Pierre Dubois, UFR de Pharmacie de Tours Philippe Maingault, Laboratoires Brothier Région Centre 26

bio-mof-1 DMASM Wavenumber (cm -1 ) Supplementary Figure S1 FTIR spectra of bio-mof-1, DMASMI, and bio-mof-1 DMASM.

bio-mof-1 DMASM Wavenumber (cm -1 ) Supplementary Figure S1 FTIR spectra of bio-mof-1, DMASMI, and bio-mof-1 DMASM. bio-mof-1 Transmittance bio-mof-1 DMASM DMASMI 2000 1500 1000 500 Wavenumber (cm -1 ) Supplementary Figure S1 FTIR spectra of bio-mof-1, DMASMI, and bio-mof-1 DMASM. Intensity (a.u.) bio-mof-1 DMASM as