Can diffuse reflectance spectroscopy emphasize skin-collagen alterations due to ageing?
Blandine Roig1, Marie Guilbert2, Anne Koenig1, Olivier Piot2, François Perraut1, Michel Manfait2, Jean-Marc
Dinten1
1
CEA, LETI, MINATEC, 17 rue des Martyrs, F38054 Grenoble, France
2
MéDIAN Biophotonique et Technologies pour la Santé, FRE CNRS 3481 MEDyC, UFR Pharmacie, 51 rue
Cognacq-Jay, 51096 Reims Cedex, France
Summary
Description of the problem: We investigate the use of spatially resolved diffuse reflectance spectroscopy for
skin ageing assessment by focusing on type I collagen as skin chromophore and scatterer.
Method of study: Measurements were carried out with a home-made dedicated probe, on the one hand, on 3D
collagen matrices, and on the other hand, on several volunteers' skin. Matrices were reconstructed using
collagen extracted from rat tail tendons. Animals of two different ages (young and old adults) were considered.
Results: In vitro acquisitions show great differences between old and young adults collagen. Analysis of the in
vivo study gives a more subtle trend. More results are consequently needed to conclude.
Conclusions: According to the in vitro results, diffuse reflectance signal of type I collagen appears as a relevant
marker for skin ageing assessment. The interpretation of the in vivo scattering spectra in comparison with in
vitro results is under investigation to complete the in vivo conclusions.
Keywords : Diffuse reflectance spectroscopy, optical properties, skin chromophores, collagen hydrogels
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Can DRS emphasize skin-collagen alterations due to ageing?
Introduction
Diffuse reflectance spectroscopy (DRS) has been widely used to determine the optical properties of biological
tissues [1]. We have developed a low-cost optical instrument based upon the spatially resolved DRS to detect in
vivo skin alterations in a clinical environment. The developed system and methodology were clinically validated
firstly on tuberculosis skin test reading [2]. Our objective is to demonstrate the potential of DRS to evaluate
type I collagen content and to detect structural alterations associated with skin ageing. In this paper, we firstly
introduce the set-up and the employed methodology. In vitro and in vivo measurements were carried out
respectively on 3D collagen gels and on skin volunteers.
Materials and methods
1. Spatially resolved diffuse reflectance spectroscopy
To assess the absorption and scattering coefficients μa and µs' of biological tissues at various wavelengths, an
optical system based on a fibers bundle has been developed for in vivo skin characterization. The easily portable
system consists of a tungsten halogen lamp as excitation source, a remote fiber optic probe for sample
illumination and backscattered light collection, a fibered spectrometer for acquisition of the reflectance spectra,
and a PC for controlling measurement in less than five seconds and for data processing. The remote probe
placed over the tissue area under examination includes a central illumination fiber and a circular network of
optical fibers located at various distances from the illumination fiber in order to collect light scattered from the
tissue at the level of the epidermis and the dermis.
The reflectance signal at a specific wavelength is measured as function of the distance between the detection
and illumination fibers. It follows an exponential decay [3]. By comparison with the values given by a Look-Up
Table previously constructed using Monte Carlo simulations or an analytical model, optical characteristics of
observed tissues are evaluated. This macroscopic information reflects the skin state: the scattering spectrum
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Can DRS emphasize skin-collagen alterations due to ageing?
permits to determine the scattering structure dimension and the absorption spectrum gives access to the amount
of the main skin chromophores (melanin, oxygenated hemoglobin, deoxygenated hemoglobin, lipid and water).
2. Extraction, preparation of ageing collagen and in vitro measurements
Native type I collagen was extracted from rat tail tendons of different-age animals (young adults of 2 months
and old adults of 2 years), lyophilized and stored at -80°C. For preparing 3D gels, lyophilized collagens were
dissolved in18 mM acetic acid at concentrations of 3 and 5 mg/ml. To generate reconstituted collagen hydrogel
constructs from extracted collagen, NaOH and other additives were added to the collagen acidic solutions
leading to neutralized collagen gel solutions at final concentrations of 1.5 and 2.5 mg/ml.
1.5ml gels were prepared in polystyrene 24-well plates and gelation of constructs was achieved by incubation at
37°C during 30 minutes. Then the probe was placed over each well for DRS measurement.
3. In vivo measurements
In vivo DRS data were also acquired on a panel of 30 volunteers’ forearms and faces of different ages from 20
to 60 years and of skin II and III phototypes.
Results
We present the potential application and relevance of the method for skin ageing assessment by focusing on
type I collagen as skin chromophore and scatterer [4]. In vitro typical raw data are shown in figure 1.
Figure 1 should be here
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Can DRS emphasize skin-collagen alterations due to ageing?
The curves shapes are significantly different between extracted collagen from 2 years- and 2 months-old rats.
The trend is conserved with varying collagen concentrations (1.5 and 2.5 mg/ml). This spectral distinction is
essentially due to differences of scattering properties of collagens between 2 months and 2 years-old rats since
the concentrations are the same. This result confirms the suitability of scattering properties of type I collagen as
an in vivo marker of skin ageing.
As far as the in vivo study is concerned, the discrimination between the youngest and oldest volunteers is not as
straightforward as for in vitro raw spectra. Therefore, the optical properties extraction method was applied to
the in vivo raw data adding type I collagen as a chromophore of interest [5]. Then, we investigate the collagen
contribution to both the absorption and scattering spectra in the 450 - 900 nm wavelength range and attempt to
quantify the content and structure changes with age.
Discussion
In vitro investigations validate the use of DRS to emphasize collagen alterations associated with age. The
contribution of type I collagen to the in vivo absorption spectrum is still under investigation. Preliminary results
seem to show a correlation between the in vivo scattering properties and ageing. Based on in vitro results, intersubject changes are being considered.
References
[1] JACQUES SL, Web site : http://omlc.ogi.edu/news/jan98/skinoptics.html (1998)
[2] KOENIG Anne et al, Diffuse reflectance spectroscopy: a clinical study of tuberculin skin tests reading,
Proc. SPIE. 8592, Biomedical Applications of Light Scattering VII 85920S (2013)
[3] FARRELL T.J. et al, A diffusion theory model of spatially resolved, steady-state diffuse reflectance for the
noninvasive determination of tissue optical properties in vivo,” Med. Phys. vol. 19 (1992) p. 879–888.
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Can DRS emphasize skin-collagen alterations due to ageing?
[4] TSENG S. et al, Noninvasive evaluation of collagen and hemoglobin contents and scattering property of in
vivo keloid scars and normal skin using diffuse reflectance spectroscopy: pilot study, Journal of biomedical
optics 17 (2012)
[5] TARONI P. et al, Absorption of collagen: effects on the estimate of breast composition and related
diagnostic implications, Journal of Biomedical Optics vol. 12(1) (2007)
Legend for figures
Figure 1: Typical raw diffuse reflectance spectra acquired on 2 months (a) and 2 years (b)-old rats' collagen
hydrogels at concentration of 2.5 mg/ml.
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Can DRS emphasize skin-collagen alterations due to ageing?
Figure 1
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