Skin Research and Technology 2010; 16: 305–310
Printed in Singapore " All rights reserved
doi: 10.1111/j.1600-0846.2010.00432.x

r 2010 John Wiley & Sons A/SSkin Research and Technology

Clinical study on the effects of a cosmetic producton dermal extracellular matrix components using ahigh-resolution multiphoton tomograph
R. Bazin1, F. Flament1, A. Colonna2, R. Le Harzic3, R. Bückle3, B. Piot1, F. Laizé1, M. Kaatz4,
K. König3 and J. W. Fluhr4,5
1

L’Oréal, Recherche, Chevilly-Larue, France, 2L’Oréal, Recherche, Aulnay-sous-Bois, France, 3Fraunhofer Institute for Biomedical Technology (IBMT),St Ingbert, Germany, 4Laboratory of Skin Physiology, Department of Dermatology, University of Jena, Germany and 5Bioskin, Berlin, Germany

Background/purpose: The aim of this study was to demonstrate the effects of selected plant extracts in a cosmetic
cream on the dermal network components after a 3-monthtreatment using an in vivo multiphoton tomographic device.
Methods: Twenty-four Caucasian women aged between 45
and 65 applied randomly a cosmetic emulsion B containing
active ingredients (soy and jasmine) twice a day on one arm
and its vehicle A (without active ingredients) on the otherarm during 3 months. Measurements were performed on theinternal side of the forearm before starting the treatment(T0), after 4 week (T4) and 12 weeks (T12) treatment.
Measurements consisted in a multi-layers acquisitions
using a multiphoton tomograph with subcellular resolution.
Optical sections (about 6 mm thick) were recorded from 0 toabout 200 mm using two different wavelengths: 760 and
820 nm. To compare the series of images and obtain an
objective quantification of the signal of second harmonic
generation (SHG) and autofluorescence, the method usedconsisted in taking the integrated brightness of an image(same rectangular area for all images) as a measure of thesignal. Following this step a ratio between brightness ofimages from the area treated with cream A or B and brightness of untreated area was calculated and used as an
assessment of treatment efficacy. The parameter used forstatistical analysis (variance analysis) is the difference
before and after 12 weeks of treatment by either cream A
or B of the signal ratios calculated in the upper dermis (118–
130 mm) and those from a deeper region of the upper dermis(165–178 mm).

Results: Signals (autofluorescence1SHG) of extracellularmatrix do not change significantly with time (weeks 0, 4 and12) when cream A (vehicle with no active ingredient) is
applied. Treatment with cream B results in an enhancementin the signal level of extracellular matrix at week 12. The
comparison of signals, in both areas (118–130 mm and 160–
178 mm), show an higher increase in the deeper region thanin the more superficial one for product B while we do notnotice any change with product A.
Conclusion: The multiphoton tomograph provided excellent high-resolution images, which describe clearly thedifferent skin layers, single cells and extracellular matrix
components in all the 24 volunteers. Statistic analyses
reveal a real effect for product B with selected plant extracts, known to increase collagen synthesis. Changes
observed are characteristics of modifications in dermal
collagen and elastin content. To our knowledge, it is thefirst time that it was possible to demonstrate in vivo theeffect of a cosmetic product on the superficial dermal layer,
in a non-invasive and non-destructive process, i.e. without
cutting the skin.

Mescence (AF) and second harmonic generation(SHG) can be used to perform optical imaging of
the structural components of tissues (1–9).
NIR intensities as high as a gigawatt per squarecentimeter can be applied without any damage totissues and fluorophores and deep tissue imaging
can be obtained. Multiphoton excitation occurs
only in a tiny intra-tissue focal volume of about
one tenth of femtoliter (10 ! 15 L).

excitation usingnear-infrared (NIR) femtosecond laser pulses
and high numerical aperture (NA) objectives can
induce a weak autofluorescence based on naturally
occurring endogenous fluorescent biomolecules
such as flavins, reduced nicotinamide adenine dinucleotide phosphate [NAD(P)H] coenzymes andmetal-free porphyrins as well as components oflipofuscin, collagen, elastin and keratin. AutofluorULTIPHOTON FLUOROPHORE

Key words: Multiphoton Spectroscopy – Multiphoton tomography – extracellular matrix – second harmonic generation– dermal structures& 2010 John Wiley & Sons A/S
Accepted for publication 23 January 2010

305

Bazin et al.

Multiphoton microscopes are not suitable aslaboratory devices for in vivo imaging in humans.
A multiphoton tomograph has been especiallydeveloped for high-resolution imaging of thehuman skin (10, 11).
In contrast to the standard technique for determining the histopathology of mechanicallyremoved tissue, non-invasive three-dimensional(3D) optical diagnosis has the advantage of (1)providing a painless diagnosis method without
tissue removal, (2) rapid access to information, (3)examination under natural physiological (in vivo)
conditions and (4) the possibility of long-term
studies on the same tissue area including in vivo
drug screening.
The aim of this study was to demonstrate thepotential of this technique to assess the effects
of selected plant extracts in a cosmetic cream oncollected AF and SHG signals by performing andanalyzing images of the epidermal and dermal
structures.
Here, we report a novel application of the femtosecond laser scanning system to non-invasive
high-resolution multiphoton tomography ofin vivo human skin with the capability to study
the effects of a cosmetic product with submicron
spatial resolution.

Material and Methods
The multiphoton tomograph DermaInspect device,as shown in Figs 1 and 2, consists of a compact,solid-state, turnkey, modelocked 80 MHz titanium sapphire laser MaiTai (Spectra Physics,
Mountain View, USA) with a tuning range of
750–850 nm coupled into a scanning module witha fast x,y galvoscanner, piezodriven # 40 focusing optics with high NA 1.3 (oil) and 200 mm

Fig. 1. Multiphoton tomograph DermaInspect device.

306

Fig. 2. Optical sections have been performed at different tissue depths
ranging from 0 mm (surface) down to about 200 mm on the inner side of
the forearm, on the upper the knee and on the face.

working distance. A fast PMT detector moduleand a control unit including JenLab Image software (JenLab GmbH, Jena, Germany) for image
processing were used.
Investigations on the human skin using Multiphoton tomography with a subcellular resolution
were performed in 24 women aged between 45
and 65 years who applied a test cosmetic cream
twice a day. The study was carried out after
signed informed consent was given by all participants and was approved by the local ethic
committee. Measurements were performed on
the internal side of the forearm before starting
the treatment (T0) and after a 4-week (T4) and a
12-week (T12) treatment. Additional measurements were performed on the face and on theupper level of the knee (Fig. 3, untreated areas).
Multiphoton tomography was performed at
the Clinic of Dermatology, Friedrich Schiller Universität (FSU), Jena, between June 2005 andNovember 2005.
Optical sections (5000 in total) were recordedon the internal side of the forearm (all volunteers), on the upper level of the knee and on theface (in two volunteers) as shown in Fig. 3 atdifferent tissue depths ranging from 0 mm (surface) down to about 200 mm using two different
laser wavelengths: 760 and 820 nm. The acquisition of one section (5.9 m) took 7.4 s.
Measurements were carried out at T0, T4 andT12 on (i) a defined untreated site as a control,(ii) a defined site treated with test cream A

Effects of a cosmetic product on dermal ECM components

Fig. 3. Examples of high-quality images obtained at 760 and 820 nm laser wavelength excitations on forearm knee or face skin tissue.

(cosmetic emulsion without active compounds,
the vehicle) and (iii) a defined site treated withcream B (the vehicle with soy and jasmine extracts). The sites were randomly and blindlychosen. The active ingredient is a complex ofthree active components from two selected extracts (soy and jasmine) known to contribute tocollagen and decorin synthesis.
We used a laser wavelength of 760 nm to obtaina two-photon luminescence (fluorescence, SHG)
signal of all the cells and of extracellular matrix(ECM) components with a submicron lateral
resolution and a 1–3 mm axial resolution. One
can observe that the different characteristic structures of the stratum corneum, including hexagonal-shaped cells, are found at a depth from 0 to
15 mm. When focusing deeper (Fig. 3), the signal
within the stratum granulosum and within thestratum spinosum is determined by mitochondrial
NAD(P)H fluorescence. The fluorescence isbrighter and influenced by melanin emission
from the stratum basale.
Interestingly, the ECM elements could be imaged with a high signal-to-noise ratio in all thevolunteers up to a depth determined by theworking distance of the focusing optics of
200 mm. The mean laser power was varied from12 to 49 mW to record efficient AF signals depending on the intra-tissue focal spot.
The second measurement series was performed at a laser wavelength of 820 nm. At thiswavelength, the signal is mainly produced bycollagen-related SHG and elastin-related AF. The
SHG signals produced by collagen consist ofradiation at half the incident laser wavelength.

Examples of high-quality images obtained arepresented in Fig. 3. Whereas at 760 nm laser
wavelength excitation, one can observe the stratum spinosum of the epidermis at a depth of
35 mm, deeper sections of the tissue can bereached when using an 820 nm laser wavelength:
images of elastin and collagen in the dermis arethus obtained from the forearm, the knee or theface.

Statistical Processing
A statistical approach of product effects withtime was carried out. The objective was to compare the amount of recorded luminescence signal
in the dermis at an 820 nm laser wavelength incream A- (cosmetic emulsion without active
compounds, the vehicle) vs. cream B (the vehicle
with soy and jasmine extracts)-treated skin areas
at T0, T4 and T12, at the same depth and the same
excitation laser power. To compare the series ofimages and to obtain an objective quantificationof the signal of SHG and AF, the method usedconsisted of taking the integrated brightness ofan image (same rectangular area for all images)
as a measure of the signal. The integrated brightness was defined as the sum of pixels weighted
over thevalues (or gray values) of theP brightness
%
i
N
;
where
Ni is the number of pixels
pixel 255ii¼0with gray value i.
Thus, the more intense the SHG and AF signals,
the higher the integrated brightness, and, when
there is no signal, the image is dark, i.e., all pixels
have 0 grayness and integrated brightness is also 0.

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Bazin et al.

The normalization of brightness was carriedout for tomography images in the dermis at a
fixed depth beginning at 88.5 mm from the surface
just under the stratum basale of volunteers (average value over several volunteers deduced fromthe images) up to 180 mm. As a measurement of
the effect of the treatment, the ratio of the brightness of images from the area treated with cream
A or B to that of images from the untreated area
was used. The following notations were used:& R, A, B – signal from untreated (control) skin,product A-treated skin and product B-treatedskin, respectively,& week0, week4, week12 – treatment period,& volunteer.N – volunteer number N,& depth – skin depths considered: 88–178 mm,& RP!weeks , average value of the control skinsignals for the three measurement periods:
week0, week4, week12.

Between 88 and 118 m, due to the automation of
the quantification process, it is possible to take
into account some epidermis information likeflattening of the dermal–epidermal junction.
Hence, to restrict our conclusion on dermis modification, we chose to carry out statistical analysis
between 118 and 178 m.
A one-way variance analysis (a 5 0.05) wasused to make an inference on the statisticalsignificance of difference before and after T12
weeks of treatment by cream A and cream B
signal ratios calculated in the upper dermis L2(118–130 m) and those from a deeper region of theupper dermis L1 (165–178 m).

given in Table 1 for product A and product B.
Adepth;week;P volunteers
ð3Þ
PR
depth;week;

volunteers

andBdepth;week;P volunteers
PR
depth;week;

ð4Þ

volunteers

The evolution of the ratios for product A andproduct B at T0, T4 and T12 weeks is summarized
in Figs 4 and 5, respectively.
The bars on the left side of the curves show thevalue of variance – one value of variance per line.
The error bars on the right side of the curves
denote the experimental error.
The high level of variance is mainly due tomovement artifacts of the in vivo measurements.
In this respect, the results presented above reduce
the set of data by excluding the signal values that
TABLE 1. Summary of variance analysis results on the difference in theratios of SHG and AF signals before and after 12 weeks of treatment
showing a significant difference on the L1 region after a 3-month productapplication

Region of interest
L2
118 to 130 m
L1
160 to 178 m

Effect of active
ingredients ondermal components*

F value**

P value***

0.29

1.07

3.10 ! 1

0.63

14.61

10 ! 6*This parameter is the difference between the difference of ratios before(T0) and after treatment (T12weeks) for product A and product B.
**The F value is obtained from one-way variance analysis.
***P value is significant when o5 # 10 ! 2.

Results
The ratio of the signal for cream A-treated skin isdefined as
Aweek12; depth; volunteer
ð1Þ
Rdepth;volunteer;P weeks

and for cream B-treated skin as
Bweek12; depth; volunteer
PR
depth;volunteer;

weeks

ð2Þ

The signal of untreated skin serves as a control.
To quantify the effects of the treatment averaged for all the volunteers, the ratios should beconsidered as in Eqs (3) and (4). The ratios of the
AF and SHG signals and the significant difference after 3 months of product application are

308

Fig. 4. Changes in the ratios of second harmonic generation (SHG)
and autofluorescence (AF) signals resulting from treatment withcream A. (ratios at T0, T4 and T12 weeks averaged for all thevolunteers). The error bars on the right side of curves denote theexperimental error.

Effects of a cosmetic product on dermal ECM components

Fig. 5. Changes in the ratios of second harmonic generation (SHG)
and autofluorescence (AF) signals resulting from treatment withcream B. (ratios at T0, T4 and T12 weeks averaged for all thevolunteers). The error bars on the right side of the curves denote theexperimental error.

deviate about threefold from the mean values(exclusion of 12%).
We notice that the signals (AF1SHG) of ECM
do not change significantly with time (weeks 0, 4
and 12) where cream A (vehicle with no active
compound) is applied. Treatment with cream B
results in an enhancement in the signal level of
ECM at week 12.
The results of the comparison of signals in L1
and L2 regions for both products A and B aresummarized in Table 1. The parameter used is thedifference between T0 and T12 weeks of the ratio
for product A and the same difference for ratio for
product B. This parameter represents the effect ofactive ingredients on dermal components.

Discussion
To our knowledge, it is the first time that multiphotonic tomography is used to study in vivo theeffects of cosmetic products on the skin structures.
The results clearly demonstrate that the level ofglobal signals detected in the dermis is higher after
treatment with cream B, whereas no significant
alteration is noticed following treatment with vehicle (cream A). This suggests that cream B may
have induced changes in the structure and/or thecomposition of the components of dermal tissue.
Dermal collagen and elastin content are modified in aged skin as well as in pathological skinconditions: skin aging is associated with an impaired collagen and elastin fibrillar structure. In
photoaging, for instance, this decrease is explained
mainly by the UV-induced expression of matrix

metalloproteases (MMP) that digest dermal collagen (12, 13). The second important hallmark ofskin photoaging is the extensive degradation or thesignificant alteration of the elastic fiber network,
which might also be caused by MMPs. The socalled elastosis, the deposit of truncated elastotic
material, is thought to be caused by the direct
impact of UV on the synthesis of elastic fibers.
Here, data acquisitions have been carried out on
the internal side of the forearm where photoaging
is weak, in order to focus our conclusion only on
chronological skin aging.
ECM modifications linked to aging have beenshown to be prevalent in the superficial dermal
layer (14, 15). Hence, we carried out our statisticalanalysis in the upper part of the dermal layer.
Statistical analysis of data showed that, after 3month applications of product B, the level ofsignal increases in the upper part of the dermal
layer from 118 to 178 mm depth. Interestingly, thisdifference is significant from 165 to 178 mm,
where measurement takes place in the upper
dermis close to the region of transition between
the papillary dermis and the reticular dermis.
An important parameter of skin aging is thedermal collagen content. Collagen I accounts for
70–90% of dermal ECM and its content decreases
by about 1% per year. Recently, multiphoton laser
tomography studies in the human dermis of 18
volunteers of different ages have been shown towell suited to characterize human dermis age (16).
In our study, the AF and SHG signals were notrecorded separately, but the fact that the global
signal had increased after the 3-month treatment
with cream B suggests that some changes haveoccurred in the composition of the collagen fibers
and/or the elastin in terms of structure, proportionor density. Cream B is known to enhance collagensynthesis; hence, we suspect that this signal increase is mainly linked to collagen synthesis. To
demonstrate this hypothesis, additional studies arein progress, taking into account the evolution of
the DermaInspect system, which now allow toseparately measure the SHG and AF signals (14).
Hence, we will be able to quantitatively determine
the ratio between SHG and AF signals and characterize its modification after cosmetic treatment.

Conclusion
The DermaInspect device provided excellent
high-resolution multiphoton images, which309

Bazin et al.

show clearly the different skin layers, single cells
and ECM components in all the 24 volunteers.
Interestingly, the signal depth could be enhanced down to about 200 mm and was limited
due to the working distance of the focusing
optics. Statistic analyses reveal a real effect for
product B with selected plant extracts known toincrease collagen synthesis. The changes observed are characteristics of modifications indermal collagen and elastin content. Further
studies are in progress to quantitatively evaluate
the alterations in skin composition over time andthe impact of test plant extracts on collagen andelastin fibers separately.

Acknowledgements
We would like to acknowledge Dr Ispiryan forthe statistical analysis.

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Address:
Roland Bazin
L’Oréal recherche
188, Rue Paul Hochard
94550 Chevilly Larue
Francee-mail: rbazin@rd.loreal.com