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Vibrational Spectroscopy
journal homepage: www.elsevier.com/locate/vibspec

An in vitro Raman study on compositional correlations of lipids and protein

with animal tissue hydration
Shan Yanga,*, Chirantan Senb,1, Raven Thompsona,1, Jian-Ge Zhoua, Ozan Akkusc,d,e,f
a
Department of Chemistry, Physics and Atmospheric Science, Jackson State University, Jackson, MS, 39217, USA
b
Department of Electrical and Computer Engineering, Mississippi State University, Starkville, MS, 39762, USA
c
Department of Mechanical and Aerospace Engineering, Case Western Reserve University, Cleveland, OH 44106, USA
d
Center for Applied Raman Spectroscopy, Case Western Reserve University, Cleveland, OH 44106, USA
e
Department of Orthopaedics, Case Western Reserve University, Cleveland, OH 44106, USA
f
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA

A R T I C LE I N FO A B S T R A C T

Keywords: Raman spectroscopy is a powerful non-invasive tool for detection and classification of chemical composition of
Raman spectroscopy materials including biological tissues. In this work, we report an in vitro Raman study on animal skin samples
NIR with a focus on high-frequency vibrations such as symmetric CH3 stretching mode at 2934 cm−1, and the
Skin hydration symmetric CH2 vibration mode at 2854 cm−1, OH stretching modes near 3412 cm−1, and bounded OH mode
Water probing
near 3284 cm−1. Raman data was acquired with a customized InGaAs based Raman spectrometer that con-
Protein
solidates the NIR (866 nm) light and the InGaAs detector and is particularly suitable for probing high-frequency
Lipids
vibrations. The Raman spectra of fat, tendon, and muscle tissues are also analyzed to determine the spectroscopic
identities of CH and OH groups in skin. Our results suggest that the protein is beneficial for the maintenance of
skin hydration, as it has higher water capacity and greater capability to retain water than lipids. This conclusion
is consistent with the additional discovery that water exists in fat mainly as unbound type, while part of water
exists as bound type in muscle.

1. Introduction However, FTIR measurement on skin can only be performed in reflec-

Dermal hydration is critical to maintain skin homeostasis and in-
tegrity. Skin hydration affects not only the appearance of the skin but
also the normal function of skin as a protection barrier [1,2]. Reduction
of the water level of skin can prevent normal desquamation leading to
dryness, roughness, scaling, and flaking of skin [3]. Skin hydration is
also found to be the most important external factor responsible for
optimal wound healing [4]. The hydration level in human skin may be
evaluated by measuring the electric parameters (e.g., conductivity, re-
sistivity, dielectric constant) of the skin [5–7]. The results of these
measurements may be affected by topical ingredients or the actual
surface variation of contact between the skin and sensor/transducer
[8,9].
Optical probing of hydration level in skin has a unique advantage as
it is non-invasive and requires no contact with skin [10–17]. Fourier-
transform infrared spectroscopy (FTIR) is sensitive to water compo-
nents and thus has been used extensively in this regard [11–13].
tion mode, which requires a close contact of the sample with the probe.
The difficulty to maintain a constant pressure between the soft skin
tissue and the probe makes quantitative analysis a challenge. Raman
spectroscopy is another optical technology suitable for analysis of skin
contents. Raman analysis on skin hydration emerged over two decades
ago, spectral features in the high-frequency region were absent from
those early studies due to dropped sensitivity of the detectors [18–20].
Raman spectra of skin showing clear water profile and detailed CH
structures were later reported by Caspers using confocal Raman spec-
troscopy [21,22]. Recently, Choe et. al. deconvoluted the CH stretching
band of lipid-keratin in the range of 2820–3030 cm−1 and OH
stretching band of water in the range of 3350–3550 cm−1, and studied
the profile variation of bound water affected by protein and lipids in-
teractions at different depths [23]. Quatela et. al. observed variation of
spectral markers including OH and CH groups among different in-
dividuals [16]. In this work, we report a study on the discrepancy of
skin hydration on the surface and its correlations with compositional

⁎
Corresponding author.
E-mail address: Shan.Yang@jsums.edu (S. Yang).
1
Undergraduate Researcher.

https://doi.org/10.1016/j.vibspec.2020.103022
Received 24 September 2019; Received in revised form 31 December 2019; Accepted 11 January 2020
S. Yang, et al.
variations of protein and lipids.
The Raman spectroscopy study was conducted by a customized
Raman spectroscopy based on an Indium Gallium Arsenide (InGaAs)
detector (sensitive to short wave infrared in the range of. 0.9–1.7 μm)
and NIR light operates at 866 nm. The combination of the detector and
the laser yielded the advantages of high quantum efficiency and re-
duced fluorescence interference. The InGaAs detector was integrated
into a transmissive volume phase holographic (VPH) diffraction grating
based spectrograph. The high dispersion (which leads to high numeric
aperture) and high optical throughput of the VPH gratings enabled the
high efficiency of the spectrograph even at a compact size. The results
from the animal skins indicate that the customized InGaAs Raman
system can not only probe the level of hydration in water skin, it can
further qualitatively evaluate the compositional variations associated
with protein and lipids.
2. Materials and methods
2.1. Samples
The chicken skin, muscle, and tendon tissues were removed from
chicken wings purchased from local supermarkets, while the chicken fat
tissues were obtained from chicken thighs. Raman measurements were
performed over those tissues that were either cut in small pieces
(< 5 mm in any dimension), or from the cross-sections of chicken wings
that were cut in half. The pork skin, muscle and fat tissues were cut
from the pork belly/back meat that was also purchased from local su-
permarkets. The samples were purchased within 48 h after their dis-
tribution to the supermarket from local farmers, samples were stored in
refrigerator unless they were intentionally left outside for dehydration.
Samples were wiped with paper towels to remove the water on the
surface ∼10 min before experiments. Several samples were covered
with quartz plates (1 mm thickness) to reduce dehydration during the
experiments, while the rest of the samples were directly examined. The
dehydrated data was taken from samples after 6−72 hours of air drying
at room temperature or overnight oven drying (in tubes with open
ends) at 45 °C.
2.2. Raman spectrometer
The Raman spectrometer was a customized dual-wavelength ex-
citation system that operates at 866 nm or 1064 and has been described
elsewhere [24]. Briefly, The spectra were recorded with an InGaAs
array camera (Du490A-1.7, Andor) mounted to a compact volume
phase holographic transmission grating based spectrograph (Wasatch
Photonics). The spectrograph covers a fixed range of 1092–1345 nm,
corresponding to Raman shifts of 2390-4110 cm−1 under 866 nm ex-
citation from tunable Ti:Sapphire laser (3900S, Spectra Physics). The
laser power was adjusted to ∼ 85 mW on the sample surface. A short
pass filter (Thorlabs FESH1000) was used to clean the laser. A dichroic
beamsplitter (Di02-R1064−25 × 36, Semrock) was used to separate
the laser and Raman signals. A long-pass filter (FELH1100, Thorlabs)
was used to remove the Rayleigh scattering of the laser lights. The
objective turret mounted with three different objective lens allows
switching between lenses with magnification power of 10, 40, or 100.
The laser spot focused on the sample surface was estimated to be less
than 4 μm in diameter and < 8 μm in depth. The integration time for
the representative spectra was 30 s and averaged 6 times. No smoothing
was applied to the acquired Raman spectra. The fluorescence back-
ground of the Raman spectra from all samples was removed by sub-
tracting a fluorescence baseline from the raw data. The baseline is
constructed by linear piecewise segments connecting multiple local
minima of the spectra, as shown in Fig. 1.
The linear regression was performed using the linear fitting function
provided by the data analysis software- OriginPro. The least sum of
squares was used to determine the best-fitted parameters. The R-
Fig. 1. Fluorescence background subtraction for Raman spectrum. A baseline
(red curve) is constructed by linear piecewise segments connecting multiple
local minima and subtracted from the raw data (black curve) to obtain the
baseline corrected spectrum (blue curve).
squared value and the probability value were automatically produced
by the software after the fitting.
3. Results
The investigation of the hydration in chicken skin tissue indicated
that the distribution of water was not uniform; most spots tested could
be categorized into two types, referred to as low-water spots and high-
water spots by the Raman signal intensity at the OH region (Fig. 2). The
categorization could also be sorted by the intensity of three CH Raman
signals located near 2854, 2895 and 2934 cm−1. The low-water spots
had rising left shoulder at 2854 cm−1, in contrast to the rising right
shoulder at 2934 cm−1 for high-water spots. In the OH region, Raman
spectra from all spots showed profiles similar to the one from distilled
water (Fig. 2a and b), whose intensity was scaled down for better re-
ference. All Raman spectra shown in Fig. 2 were normalized according
to the CH signal located at the middle (∼2895 cm−1) of the two wings,
while the water intensity distribution of 66 spots were normalized ac-
cording to both CH signals at the middle (∼2895 cm−1) and the right
(∼2934 cm−1), as shown Fig. 2c. The 66 sampling spots were randomly
selected from a dozen skin pieces that were removed from various lo-
cations of 5 different chicken wings. The intensity ratio of the two CH
signals, represented by I2934/I2895, was used as the horizontal axis im-
plying the compositional variations at those 66 spots. The distribution
plot indicated a strong correlation between the OH intensity (normal-
ized against the CH signal at 2895 cm−1) and the protein concentration
(R2 = 0.95, p < 0.001), as shown in Fig. 2c.
After 24 -h air drying at room temperature, Raman signals in the OH
region decreased significantly at high-water spots and almost com-
pletely disappeared at low-water spots, as shown in Fig. 2. The spectra
shown were acquired from the same piece of chicken skin removed
from a chicken wing and the experiment was repeated at multiple spots
of varied samples. Such variation was repeatable over a dozen other
skin tissues. The rate of dehydration depends on the size of the skin
pieces; the time of dehydration is approximately tripled to observe si-
milar effects from skin tissues that were kept on a wing. The high-water
spots were found to hold water longer than surrounding low-water
spots. Exploring various spots on the skin samples showed that the
majority of sampled spots were low-water type (Fig. 2c), and high-
water spots were rare. Some spots show mixed profiles of low and high-
water spots, i.e., both CH shoulders are partially raised, these spots
typically had the I2934/I2895 ratio close to 1.0 (Fig. 2c). The last spec-
trum from each individual skin piece was taken within 15 min of the
acquisition of the first spectrum. The hydration reduction due to air
drying in this 15 min was negligible as no difference was observed
when the skin is covered with a quartz slide.
S. Yang, et al.
Fig. 2. Raman spectra of chicken skin at representative (a) low-water spots and
(b) high-water spots before (blue curve) and after 24 -h air drying (red curve).
Peaks marked with vertical dashed lines from low to high were located at 2854,
2895, and 2934 cm−1 respectively. The spectra were acquired with 85 mw,
866 nm laser light with 30 s exposure and 6 averages. All skin spectra were
normalized according to the 2895 cm−1 signal for easier comparison, while the
spectrum of distilled water was rescaled to match the corresponding OH signal
of the skin. (c) Water intensity distribution of 66 skin spots from 12 skin
samples removed from random locations of 5 different chicken wings. The
water intensities (OH signal at 3412 cm−1) were normalized against the CH
signals at 2895 cm−1 (blue triangles) and 2934 cm−1 (red dots) correspond-
ingly. The blue straingtline is the linear regression of the scattered data points
represented by the triangles. The intensity ratio of the two CH signals is used as
horizontal variable I2934/I2895 to indicate a variation of protein concentration.
The data were taken under the condition of 85 mw, 866 nm laser illumination
with 30 s single scan.
Raman spectra from different chicken tissues including skin (with
both types of spots), muscle, tendon, and fat were acquired for com-
parison (Fig. 3). These spectra were acquired under the same experi-
mental conditions, and the sample tissues were cut in similar thickness
except the tendon tissue (it was peeled off from somewhere between a
bone and a muscle) which was partial transparent and thin like a paper
sheet. The fat tissue was cut from a chicken thigh, while all other tissues
were cut from chicken wings. These tissues showed different pattern of
three major CH signals, however, the correlation between the two CH
wings and the detected water intensity (without dehydration) was
consistent with the observations over the skin tissues, i.e., higher left
shoulder associated with reduced OH intensities, while higher right
shoulder associated with enhanced OH signals, details were presented
in Fig. 3a.
Vertically zoomed Raman spectra of dehydrated chicken muscle,
tendon, and fat, together with that of nondehydrated fat were presented
in Fig. 3b. All spectra were normalized according to the CH signal at
2895 cm−1. The dehydrated samples were prepared by 3-day air drying
at room temperature or overnight oven drying at 45 °C. The Raman
Fig. 3. Raman spectra of chicken tissues (a) before and (b) after dehydration
(except fat). Spectra in (b) were vertically enlarged. Dehydrated fat was pre-
pared with 6 h of air drying at room temperature, while dehydrated muscle or
tendon were prepared with 72 h of air drying at room temperature or overnight
oven drying at 45 °C. All spectra were acquired under the condition of 85 mW
laser at 866 nm illumination, with 30 s exposure, and averaged 6 times. All
spectra in (a) and (b) were normalized according to the 2895 cm−1 signal.
spectrum of the fat showed weak signals in the OH region before de-
hydration, the weak signals disappeared after 6 h of air drying at room
temperature. Additional Raman peaks emerged from muscle samples
after dehydration. Specifically, the oven-dried sample showed a Raman
signal at 3284 cm−1, while the air-dried sample showed a signal that
was slightly shifted to ∼3300 cm−1 and another weaker signal at
3421 cm−1.
The correlation between the raised CH shoulder and the OH signal
intensity was also observed in the Raman spectra of pork tissues, as
shown in Fig. 4a. Comparing to Raman spectra of chicken tissues, two
differences were observed from the spectra of pork tissues. First, the
middle CH signal shifted to 2886 cm−1. Second, low-water spots were
never observed from pork skin, as all spectra had their right CH
shoulder at ∼2936−2939 cm−1 raised. Normalized intensity dis-
tribution of 25 random points over three skin pieces (two belly skin
pieces and one back skin piece, all at sizes of ∼1 × 1 cm2) was shown
in Fig. 4b. The OH intensity from these spots were not uniform, how-
ever, the ratios of OH signal at 3412 cm−1 to the CH signal at
2936 cm−1 were consistent (mostly < 5 % off from the average, e.g.,
data represented by stars in Fig. 4b) within each individual skin piece.
The distribution plot also suggested the OH intensity (normalized
against CH signal at 2886 cm−1) and the protein concentration was
correlated (R2 = 0.32, p = 0.004) based on all 25 data points (data in
blue, Fig. 4b).
4. Discussion
Raman spectra from chicken skin indicate that the water distribu-
tion and retention are non-uniform and depend on the variations of the
tissue components. Specifically, the component associated with the
Raman signal at 2934 cm−1 appears to be a positive factor for water
storing while the component associated with the signal at 2854 cm−1
plays a negative role. Further comparison of Raman spectra among skin,
muscle and fat tissues suggest that the component represented by the
2934 cm−1 signal is rich in muscle, while the component associated
with the 2854 cm−1 signal is rich in fat (Fig. 3a). This coincides with
earlier studies showing that the 2934 cm−1 peak is dominant in protein
(keratin) while the 2854 cm−1 peak is dominant in lipids 16, and they
are assigned to the symmetric CH3 stretching mode, and the symmetric
CH2 vibration, respectively [22 ,25]. Therefore, we suggest the high-
water spots on chicken skin are protein-rich while the low-water spots
S. Yang, et al.
Fig. 4. (a) Normalized Raman spectra of pork tissues including skin, muscle,
and fat. A spectrum of distilled water was rescaled for reference. Spectra from
tissues were acquired under 85 mW laser excitation at 866 nm. The exposure
time was 30 s with 6 averages. (b) Water intensity distribution of 25 surface
spots acquired from three different pieces of skin (two pieces of belly skin and
one piece of back skin, represented by squares, stars, and dots respectively). The
intensity normalization followed the same procedure as in Fig. 2c. The blue
data spots were normalized against the middle CH signal at ∼2886 cm−1, and
the red data were normalized against the right CH shoulder at 2936 cm−1. The
linear regression was based on all 25 blue spots.
are lipids-rich. This suggestion is supported by the observation that the
muscle tissue has the highest OH (3412 cm−1) to CH (2895 cm−1)
signal intensity ratio, while the fat tissue has the lowest one among
those tissues under investigation. It is notable that, the OH intensity
from fat is also far less than other tissues before normalization.
Therefore, our results strongly suggest that the protein component has a
greater water retention capacity than lipids.
The dramatic intensity reduction of Raman signals (e.g., ratio of the
OH signal at 3412 to CH signal at 2895 cm−1 in muscle dropped from
1.4 to 0.2 after dehydration) in the 3100−3700 cm−1 range after air
drying at room temperature indicate preferential evaporation of un-
bound or loosely bound water (Fig. 3), as suggested in earlier studies 14.
The shape of the Raman signals (Fig. 3b) from dehydrated muscle in-
dicates the residues are no-longer due to free water, the emerged peak
at ∼3300 cm−1 is probably due to water that is bounded to the tissue
matrix and is hard to remove. While the appearance of the Raman peak
at ∼3284 cm−1 for the oven-dried muscle accompanied by the dis-
appearance of the 3300 cm−1 peak suggests that the ∼3284 cm−1 peak
is likely due to chemical bonds within the tissue. We tentatively as-
signed this peak to the NH vibration of proteins, as it is the only non
eOH vibration reported in this range. Similarly, the appearance of the
band located at ∼3421 cm−1 after air-drying suggests this signal is also
due to bound water in the muscle. The observation of water being
undetectable from 6-h of air drying fat suggests that the fat is less re-
sistant to dehydration than muscle (Fig. 3b), which may be attributed to
its lack of water-binding capability (unlike protein).
Representative Raman spectra are normalized according to the
middle of the three CH signals (2886−2895 cm−1) rather than the right
CH shoulder (2934−2938 cm−1 signal) as in earlier studies conducted
by Caspers [21,22]. Our selection is based on the interest of in-
vestigating the correlation between skin hydration with skin composi-
tions. A reference of 2934−2938 cm−1 signal, whose intensity is al-
most negligible on the fat spectrum, will conceal the contribution from
fat especially when the fat concentration is low. This can be concluded
from the intensity distribution plots shown in Fig. 2c. Among high-
water spots from chicken skin (e.g., data points whose I2934/I2895 is
greater than 1.4) and all spots from pork skin, the OH intensity nor-
malized against I2934 does not correlate with the protein concentration
variation, i.e., the influence of fat concentration variation is dis-
regarded. On the other hand, the normalization against the middle CH
signal (blue curve, Fig. 2c) shows increases in OH intensity with reduces
of fat concentration. At low water spots where fat is dominant, no
difference was observed between the two ways of normalization.
Compositional variations of lipids and protein appears more con-
sistent on pork skin, as evidenced by the smaller fluctuation of the
range of the three ratios (Fig. 4b). The narrow range of intensity var-
iation of pork skin amplifies the intra-sample differences of the average
intensities (e.g., data in squares vs data in dots, Fig. 4b), which differs
about 20 %. Such discrepancy may be due to the non-uniformity of
certain composition (e.g. cholesterol or triacylglycerols) in the skin that
also has a small contribution to the CH signal at 2934 cm− 1 (raised
right shoulder) [25]. However, the normalized intensity distribution
still supports the claim that higher protein concentration (e.g. blue
dots) correlates (p = 0.004) with increased hydration, even though the
confidence is reduced due to the smaller data range.
5. Summary
This work is an addition to the Raman studies addressing the high-
frequency vibrations in the CH and OH region of skin. The combination
of the NIR light at 866 nm and the InGaAs detector allows the ob-
servation of these high-frequency with details without fluorescence
interference. The spatial survey of CH and OH Raman region over the
skin surface indicates the protein component is positively correlated
with skin hydration, while the fat component acts the other way. This
discovery would be beneficial for research related to controlling or
improving hydration conditions in skin or other biological tissues.
CRediT authorship contribution statement
Shan Yang: Conceptualization, Methodology, Writing - original
draft, Writing - review & editing. Chirantan Sen: Writing - original
draft. Raven Thompson: Investigation. Jian-Ge Zhou:
Conceptualization. Ozan Akkus: Writing - review & editing.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest related to
this article.
Acknowledgments
This work was supported by the National Institute of General
Medical Sciences (NIGMS) and National Institute of Dental and
Craniofacial Research (NIDCR) of the National Institutes of Health
(NIH) under award number SC2DE027240, and the Mississippi INBRE,
funded by an Institutional Development Award (IDeA) from the NIGMS
of the NIH under grant number P20GM103476. The equipment was
partially supported by the National Science Foundation (NSF) under
award number 1332444.
S. Yang, et al.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.vibspec.2020.103022.
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