In Vivo Raman Measurements of Human Skin: 1. Sample
In Vivo Raman Measurements of Human Skin: 1. Sample
some tape were used to ensure the fingers Figure 2: Top: example of the intensity
remain still. We checked that the measurement response curve of the Silicon band.(521cm-1)
is repeatable by recording five successive for the determination of the depth of focus.
spectra of the CH/OH region on the surface of Bottom: FWMH value depending on system
the sample. This showed a good focus stability parameters.
with band area fluctuation of less than 5%.
Results
3. Measurement Conditions
Two different objectives were used depending 1. Choice of the excitation wavelength:
on the wavelength range: 20X LWD NIR spectra recorded at 633nm, 785nm and
(N.A=0.40) and 50X LWD visible (N.A=0.55). 830nm
The laser power available at the sampling point
We recorded the Raman spectra of the surface
is around 5-10 mW, spectra were recorded at
skin at 633nm, 785nm and 830nm which are
the three wavelengths 633, 785 and 830 nm in
shown Fig.3a. The CDD response is however
a few tenth of seconds.
reduced in the high frequencies domain at
Before undertaking surface and in-depth profile
785nm and even limited to 2200cm-1 at 830nm
measurements, we determined the
which can be rather limitating when whishing to
approximate depth of focus of the objective
observe the OH, NH and CH stretching modes
used which is directly linked with the volume
lying around 2800 cm-1 up to 3500 cm-1. It is
from which Raman signal is collected. For all
then necessary to select InGaAs detectors
measurements, the confocal hole was opened
optimized for the NIR region. In this application,
at either 200 or 400 µm to find the best
the use of the readily implemented 633nm
compromise between axial resolution and
laser excitation for skin analysis is possible
signal intensity due to the limited laser power
without observing any tissue autofluorescence.
at the sampling point. The depth of focus was
However, for other in vivo applications (i.e.
roughly estimated for such a confocal hole and
study of internal organs such as liver, brain,
for both objectives (50X LWD visible and 20X
muscles…) and the investigated skin which
LWD NIR) and the results are presented below.
has undergone cosmetic pre-treatments
For the evaluation of the depth of focus, a
(hydration for example), it is sometimes
silicon sample was moved through the laser
preferable to use Near InfraRed excitations to
focus and the signal going through the
avoid interference of fluorescence emission.
confocal pinhole was measured. The axial
Moreover, powerful lasers focused on
resolution is inferred from the width of the
micrometric surfaces lead to increases in most
response curve at 50% of the maximum signal
tissue temperature4. As most tissues exhibit a
(Full Width at High Maximum). The derived
minimum absorption of light in the NIR domain5,
axial resolutions are shown below.
using excitation wavelength in this domain will
minimize the risks of sample heating and/or
100 photochemical interactions.
.u.)
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2934.8
at 1616 cm-1 seems to vanish when
penetrating deeper into the skin. The band at
2878.8
1412 cm-1 also decreases when probing
1651.8
1002.9
887.3
1446.7
1346.0
1412
1616
500 1000 1500 2000 2500 3000 3500
Wavenumber (cm-1) Intensity (counts/s)
0µm
1652
40
80
1297
1003
120
1452
1272
Intensity (a.u)
160
1033
852
935
200
240
1000 1200 1400 1600
Wavenumber (cm-1)
Table 1: Summary of major vibrational bands identified in skin: ν, stretching mode; νs, symmetric
stretch; δ, bending mode.
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broad band (see Fig. 3a, spectrum at 633nm). Figure 5 : Non hydrated skin (a) 3D view of the
is constituted of the v(OH) broad band and of spectra of the CH/OH region recorded at different
the v(NH) thinner band located around depths within the non hydrated skin. (b) In-depth
3300cm-1. profile of the relative evolution of the v(OH) band
The evolution of the water content in tissue area [3350-3550]cm-1.
can be determined by calculating the area
below a part of the complex broad band in the
range [3350-3550] cm-1 of the normalized
spectra (see Puppels et al. 1-3) 2.0
1.0
2.0
0.0
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Conclusion References
1. “In Vitro and In Vivo Raman Spectroscopy
Thanks to confocal Raman spectroscopy we
of Human skin”, P.J. Caspers, G.W
have been able to investigate in vivo the
Lucassen, R. Wolthuis, H.A. Bruining, G.J.
epidermis chemical composition in its
Puppels, Biospectroscopy, 4, S31-S39,
thickness. In depth measurements allowed us
1998.
to observe modifications of the relative
2. “In Vivo Confocal Raman
concentration of some components (water,
Microspectroscopy of the skin :
proteins) depending on the depth in the skin
Noninvasive determination of molecular
and on its state of hydration.
concentration profiles ” , P.J. Caspers,
G.W Lucassen, Elizabeth A. Carter, R.
The use of larger numerical aperture objectives
Wolthuis, H.A. Bruining, G.J. Puppels,
would be helpful to emphasize the in-depth
2001
variations because of the possible gain in axial
3. “Automated depth scanning confocal
resolution and therefore in-depth discrimination.
Raman Microspectrometer for rapid in-vivo
Indeed, the operator could then probe in depth
determination of water concentration
with a smaller step size.
profiles in human skin”, P.J. Caspers, G.W
Lucassen, H.A. Bruining, G.J. Puppels,
As a possible gain on the above
2000
measurements, the use of more powerful
4. Handbook of Raman Spectroscopy.
commercially available NIR diode lasers would
Practical spectroscopy series volume 28.
yield up to ten times more laser power at the
Ed. Ian R. Lewis and Howell G. M.
sample (60-80 mW) and would result in a
Edwards, pp549-574.
tremendous improvement in terms of signal
5. Handbook of Vibrational Spectroscopy,
collection and integration time.
vol.5, Ed. J.M. Chalmers and P.R. Griffiths,
Wiley, pp3362-3375.
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