1. Department of Dermatology and Allergy Charité - Universitätsmedizin Berlin Overview of noninvasive methods for determination of carotenoid concentrations in mammalian skin M.E. Darvin, J. Lademann Charité-Universitätsmedizin Berlin, Center of Experimental and Applied Cutaneous Physiology, Department of Dermatology, Venerology and Allergology, Berlin, Germany

2. Carotenoids are known to be powerful antioxidants acting effectively against reactive oxygen species. The ability of carotenoids to quench oxygen radicals (including singlet oxygen) is related to the conjugated carbon double-bond system, and maximum protection is provided by those having nine or more double bonds. Most prominent carotenoids being accumulated in human skin are beta-carotene (9 double bonds) and lycopene (11 double bonds) and their isomers.

3. High-performance liquid chromatography (HPLC) is a widely used „gold standard“ method for determination of carotenoids. This method is highly invasive, time-consuming and expansive. Moreover, the oxidation of carotenoids during the sample preparation cannot be excluded. For analyzing the kinetics of carotenoids in the skin noninvasive methods are irreplaceable and should be performed.

4. Carotenoids cannot be detected in the skin using fluorescence analyses because their fluorescence efficiency is very low (10 - 4 -10 -5 ). Optical methods for noninvasive determination of carotenoids in mammalian skin: 1. resonance Raman spectroscopy 2. Raman microscopy 3. reflection spectroscopy 4. skin color measurements

5. Resonance Raman spectroscopy 1 – Ar + laser; 2 – lens system; 3 – filter (488nm/514.5nm); 4, 9 – optical fiber; 5 – excitation channel; 6 – optical imaging system; 7 – skin; 8 – receiving channel; 10 – spectrometer; 11 – CCD; 2 – computer; 13 – photo detector Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013

6. Brandon et al. Cancer Prev. Res. 3(4): 529-538, 2010 Excitation wavelength 488 nm Fiber optic-based resonance Raman spectroscopy for endoscopic measurement of carotenoid oxidative breakdown in living tissue

7. Advantages: - measurement quickness - high sensitivity - selectivity between beta-carotene and lycopene - increasing the measurement accuracy using photobleaching effect - measurement stability - transportability Limitations: - influence of other carotenoids - reabsorption of Raman signal at 527.2 nm by lycopene Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013 Resonance Raman spectroscopy

8. Raman microscopy 1 – laser (NIR or VIS); 2 – short-pass filter; 3 – objective; 4 – mirrors; 5 – skin; 6 – laser rejection filter; 7 – lens; 8 – optical fiber; 9 – spectrometer; 10 – CCD; 11 – computer Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013

10. Advantages: - measurement of axial distribution of carotenoids in the skin Limitations: - low and very low carotenoid concentrations are not detectable under non-resonant excitation - bulky size - high price Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013 Raman microscopy

11. Reflectance spectroscopy 1 – LED (440 - 490 nm); 2 – skin; 3 – focusing system; 4 – replicated holographic grating spectrometer; 5 – Bluetooth Darvin et al. J. Biophotonics 5(7): 550-558, 2012

12. „Scanner“ in use

13. Advantages: - low price - measurement stability - compact size (easy to transport) - independence from electric mains Limitations: - low carotenoid concentrations are not detectable - could be applied only on thenal and plantar skin areas where the epidermis is thick enough and influence of melanin and blood chromophores is less pronounced Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013 Reflectance spectroscopy Darvin et al. J. Biophotonics 5(7): 550-558, 2012

14. Skin color measurements Bersha. Master Thesis, University of Eastern Finland, 21 June 2010 b*-value characterizing yellowness of the skin serves as a measuring parameter for epidermal carotenoids

15. Alaluf et al. Journal of Nutrition 132(3), 399-403, 2002 b*-value was found to correlate with concentration of carotenoids in human epidermis

16. Advantages: - low price - compact size (easy to transport) - independence from electric mains Limitations: - low carotenoid concentrations are not detectable - could be applied only on thenal and plantar skin areas where the epidermis is thick enough and influence of melanin and blood chromophores is less pronounced - measurement stability is not high enough Darvin et al. J. Biomed. Opt. 18(6), 061230, 2013 Skin color measurements

17. Resonance Raman spectroscopy Raman microscopySkin color measurementsReflection spectroscopy Size/weightcompact to bulkybulkycompact Transportabilityyesnoyes Pricemiddle to highhighlowlow to middle Measurement time 3 to 90 sec around 60 seconds around 30 sec around 60 sec Measurement stability better than 10% better than 10% no data better than 10% Light sourcelaser, LEDlaserlamp, LEDLED Measurement depth up to 200 µm up to 40 µm up to 200 µm up to 200 µm Selectivityto lycopeneno Electric mainsneeded don’t needed Personal rating 1 3 4 2 Conclusions Comparison of optical methods for measurement of carotenoids in human skin

18. Thank you for your attention