Received: 27 April 2022 | Revised: 7 June 2022 | Accepted: 12 July 2022

DOI: 10.1111/exd.14645

REVIEW ARTICLE

Novel aspects of Raman spectroscopy in skin research

Dominique Lunter1 | Victoria Klang2 | Dorottya Kocsis3 |

Zsófia Varga-­Medveczky3 | Szilvia Berkó4 | Franciska Erdő3,5

1
Department of Pharmaceutical
Technology, Institute of Pharmacy and Abstract
Biochemistry, Eberhard Karls University
The analytical technology of Raman spectroscopy has an almost 100-­year history.
of Tübingen, University of Tübingen,
Tübingen, Germany During this period, many modifications and developments happened in the method
2
Division of Pharmaceutical Technology like discovery of laser, improvements in optical elements and sensitivity of spectrom-
and Biopharmaceutics, Department of
Pharmaceutical Sciences, Faculty of Life
eter and also more advanced light detection systems. Many types of the innovative
Sciences, University of Vienna, Vienna, techniques appeared (e.g. Transmittance Raman spectroscopy, Coherent Raman
Austria
3
Scattering microscopy, Surface-­
Enhanced Raman scattering and Confocal Raman
Faculty of Information Technology
and Bionics, Pázmány Péter Catholic spectroscopy/microscopy). This review article gives a short description about these
University, Budapest, Hungary different Raman techniques and their possible applications. Then, a short statistical
4
Institute of Pharmaceutical Technology
part is coming about the appearance of Raman spectroscopy in the scientific litera-
and Regulatory Affairs, Faculty of
Pharmacy, University of Szeged, Szeged, ture from the beginnings to these days. The third part of the paper shows the main
Hungary
5
application options of the technique (especially confocal Raman spectroscopy) in skin
EA 6295 Nanomédicaments et
Nanosondes, University of Tours, Tours, research, including skin composition analysis, drug penetration monitoring and analy-
France sis, diagnostic utilizations in dermatology and cosmeto-­scientific applications. At the
Correspondence end, the possible role of artificial intelligence in Raman data analysis and the regula-
Franciska Erdő, Faculty of Information tory aspect of these techniques in dermatology are briefly summarized. For the future
Technology and Bionics, Pázmány Péter
Catholic University, Práter u. 50A, of Raman Spectroscopy, increasing clinical relevance and in vivo applications can be
Budapest 1083, Hungary. predicted with spreading of non-­destructive methods and appearance with the most
Email: [email protected]
advanced instruments with rapid analysis time.
Funding information
National Research, Development and KEYWORDS
Innovation Office of Hungary; Studium artificial intelligence, cosmetoscience, Raman spectroscopy, skin composition, skin diagnostics,
Loire Valley-­Institute for Advanced skin research, topical drug penetration
Studies

1 | I NTRO D U C TI O N Raman spectroscopy, fluorescence imaging and multispectral opto-

acustic tomography. All these methods have advantages and limita-
Imaging modalities help in the development of topical drugs and tion which are summarized in details by Schneider et al, 2019a and
cosmetics and also contribute to the early diagnosis, monitoring and 2019b.1,2 This review article aimed to focus on Raman spectroscopy
grading of various pathological or physiological conditions in the in skin research. This technology uses near-­infrared lasers which
skin. Different techniques are available in the toolbox of dermatolo- generate photons from the test preparation (e.g. skin tissue) that
gists and cosmeto-­scientists including dermoscopy, confocal micros- scatter at the same and at different energies at each chemical bond
copy, optical coherence tomography, high-­
frequency ultrasound, within the structure. The changes are detected by the device and

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction
in any medium, provided the original work is properly cited and is not used for commercial purposes.
© 2022 The Authors. Experimental Dermatology published by John Wiley & Sons Ltd.

Experimental Dermatology. 2022;31:1311–1329.  wileyonlinelibrary.com/journal/exd | 1311

1312 | LUNTER et al.

translated into highly specific and characteristic graphs (spectra). It medical and environmental samples7 and also for art and archeol-
can be applied both on in vivo and ex vivo/in vitro samples. The main ogy.8 Non-­destructive Raman analysis has been used for cultural
strength of Raman spectroscopy is that it is a non-­invasive, non-­ heritage diagnostics9 and to evaluate the ageing and degradation of
destructive method, which makes possible the in vivo sample analy- plenty of precious artefacts, paints and statues.10 During the last
sis and further investigation of ex vivo samples after Raman analysis. almost one century, the market for Raman spectroscopy has had
Also, the coupling of Raman spectroscopy with confocal microscope plenty of time to mature, and users are continuously working on to
enables the possibility of making axial screening. These screening find new application areas.
steps can be combined with automatic focus which enables the mea- The continuous development of the technology resulted in
surements to be made automatically. No sample preparation is re- (1) increased light transmission due to the more advanced optical
quired which simplifies the method and shortens the time collecting components, such as the lens, mirror and, especially, optical filters
the experimental results.3 for the removal of scattered light; (2) improved stability due to de-
In the current paper after some introductory words about the velopments in the spectrometer; and (3) enhanced light detection
history and principle of Raman techniques, the main types of the sensitivity with the use of detectors such as charge-­coupled devices
Raman spectroscopy in dermatological research, the most import- (CCDs).
ant application possibilities in skin analysis, and the use of machine Developments in coherent Raman scattering microscopy ap-
learning in data analysis are summarized. Finally, a short chapter plications such as stimulated Raman scattering and coherent anti-­
about the regulatory aspects is presented. Stokes Raman scattering microscopy offer exciting potential in
biological imaging applications (see later in the article). Moreover,
they offer huge potential for sub-­diffraction limited microscopic ap-
2 | H I S TO RY O F R A M A N S PEC TROS CO PY proaches such as tip-­enhanced Raman scattering (TERS), which are
now crossing boundaries of spatial resolution that have previously
The principle of Raman spectroscopy is based on the Raman effect not been accessible with conventional Raman microscopy. Another
which has been discovered more than 90 years ago. The phenom- advancement is surface-­enhanced Raman spectroscopy (SERS). It
enon of inelastic scattering of light (Raman scattering) was discov- uses nanostructured metal surfaces to generate Raman spectra that
ered by Dr. C.V. Raman (1888–­1970) in 1928. Sir Chandrasekhara are several orders of magnitude higher than regular ones.
Venkata Raman was an Indian physicist known about his work in
the field of light scattering.4 He developed a spectrograph, and to-
gether with his student, K. S. Krishnan discovered that when light 3 | TH E PR I N C I PLE O F R A M A N
traverses a transparent material, a shift is happening in the deflected S PEC TROS CO PY
light in its wavelength and frequency.5 This phenomenon is called
“modified scattering” which was subsequently termed as Raman ef- Among the methods of material science, non-­destructive vibration
fect or Raman scattering. Raman received the Nobel Prize in 1930 spectroscopy (including Raman spectrometry) is known, which is a
in Physics for this discovery, and he was the first Asian to receive a set of structural analysis methods based on the excitation of vibra-
Nobel Prize in any branch of sciences. tional energy transitions and suitable for the production of chemical
Originally, extraordinary measures were required to obtain maps also. Raman scattering is an indirect way to excite vibrational
Raman spectra due to the low sensitivity of the technique. Typically, transitions. Photons emitted by irradiated (monochromatic) high-­
the sample was held in a long tube and illuminated along its length intensity laser light undergo a change in frequency when they col-
with a beam of filtered monochromatic light generated by a gas dis- lide inelastically with a molecule. The magnitude of the frequency
charge lamp. The photons scattered by the sample were collected shift is the same as the vibration frequencies characteristic of the
through an optical flat at the end of the tube. To maximize the sen- molecules of the test substance, so that the vibrations of the test
sitivity, the sample was highly concentrated and relatively large substance can be identified from the spectra. The condition is a mo-
volumes (5 ml or more) were used. The use of Raman spectroscopy lecular structure, or a system of atoms connected by covalent bonds,
diminished when commercial infrared (IR) spectrophotometers be- as these result in a spectrum of sufficiently narrow bands to facili-
came available in the 1940s. However, the discovery of the laser in tate the identification of a given substance. Raman spectrometry
the 1960s resulted in simplified Raman spectroscopy instruments usually detects molecular vibrations in the range of 3200–­200 cm−1.
and also boosted the sensitivity of the technique. This has renewed Raman spectrometry complements infrared spectroscopy
the use of Raman spectroscopy as a common analytical technique.6 well, as both analyse the molecular vibrations of matter. The rel-
In the late 1970s, Raman spectroscopy combined with an optical ative sensitivity of the two methods is different, so it is possible
microscope was introduced and was used for microanalysis in sev- to study different molecular bonds and bond groups. There are
eral fields. Micro-­Raman spectroscopy became an important tool in several vibrational transitions that are inactive in infrared spec-
biology, especially for single-­cell studies. Now, many researchers in troscopy but active in Raman spectrometry, and vice versa. Raman
various fields use micro-­Raman spectroscopy. The application of this spectrometry is mainly concerned with the most common chro-
technology is widespread in analytical fields for industrial, biological, mophore groups, that is C-­O, C-­N , C-­S , S-­S , C-­C l bonds and C=C,
LUNTER et al. | 1313

C=O, C=N, N=O, C=S and C≡C bonds and bond groups, while may, in the easiest case, contain the intensities of the signal in one
IR spectroscopy is more sensitive to polar bonds and functional single wavenumber at each point in the x-­y-­coordinate system. In
groups. Water with an intense IR absorption spectrum shows only line scanning, the sample is illuminated along a line and the de-
a weak Raman scattering, so its presence during the measurement tector simultaneously detects the signals along this line. This is
is not disturbing. achieved either by conversion of the circular beam to a line focus
Chemical mapping is currently one of the most dynamically using a cylindrical lens or by using a point focus to scan along a
evolving areas of spectroscopy. The term “chemical mapping” re- line. 20 In global imaging, a two-­dimensional area of the sample is
fers primarily to methods based on vibrational (infrared and Raman) illuminated. To this end, the laser is defocused which in turn leads
spectrometric techniques.11 Chemical mapping techniques combine to lower intensity of the incident light at the sample. The light is
the advantageous properties of a spectrometer and a microscope. guided towards a detector comprising many thousand elements
The information carried by the vibration spectra, which is very char- that acquires all signals simultaneously. Here, each wavenumber is
acteristic of each molecule, can be combined with the possibility of detected one after the other. For imaging purposes, an area of in-
spatial mapping, so the concentration and location of the compo- terest may thus be mapped in a point-­by-­p oint approach or imaged
nents can be determined in heterogeneous systems. This method by global imaging (depending on the type of instrument used). In
is becoming increasingly important in many fields of science and point-­by-­p oint mapping, a full spectrum is acquired in each point.
industry, from the semiconductor industry,12 medicine,13 the paper While the acquisition of one spectrum is very fast, the need for
industry,14 the food industry15 and the plastics industry16 to phar- acquiring many spectra slows down the process. In global imaging,
11,17,18,19
maceutical technology. all points are illuminated at the same time but only one wavenum-
A practical advantage of Raman spectrometry is that in most ber is detected. In general, less light is guided to the detector than
cases no sample preparation is required. Another major advantage is in point-­by-­p oint mapping, thus requiring longer acquisition times.
that due to the high power of the irradiated light, it is also possible to This means that in the case that only one signal at one wavenum-
measure the samples through glass and plastic. The disadvantage of ber is of interest, this approach is faster. But, if a full spectrum
this method is that it can cause degradation in more sensitive sam- needs to be acquired, the process is slowed down substantially
ples and some substances show fluorescence, which significantly and may then be even slower than point-­by-­p oint mapping.
impairs the signal-­to-­noise ratio.

4.2 | Transmittance Raman spectroscopy

4 | R A M A N S PEC TROS CO PI C
TEC H N I Q U E S In transmittance Raman spectroscopy, the whole sample or a large
part of it (typically several mm) is illuminated and the transmitted
Raman spectroscopic investigation of the skin, changes in skin light is detected. The detected spectrum contains an average spec-
composition due to age or diseases, or analysis of drug delivery to trum of the sample that it diffused through. It is thus not useful for
the skin are mostly done by either acquiring spectra along a line generating spatially resolved information like it is aimed, for example
perpendicular to the skin surface or by two-­dimensional imaging. in skin penetration research. If alterations in the overall spectrum
The former gives penetration depth profiles similar to those ob- are high enough, it may still be used, for example for quick detec-
tained by conventional methods such as tape stripping. The latter tion of cancerous tissue. The main area of application remains non-­
enables the visualization of drug (or other substances) distribution destructive quantitative analysis of dosage forms like ointments,
in the skin. tablets or capsules. 21

4.1 | Types of data acquisition 4.3 | Coherent Raman scattering microscopies

In general, spectra may be acquired in a point-­by-­p oint mapping Coherent Raman spectroscopy comprises two techniques: coher-
(using a spot focus), line scanning (using a line focus) or global ent anti-­Stokes Raman scattering (CARS) and stimulated Raman
imaging approach (using wide-­field illumination). Point-­by-­p oint scattering (SRS). Both are stimulated and thus need two lasers:
mapping is the most frequently used approach. Here, spectra “pump” and “Stokes.” The frequency difference between the two
are acquired in each point in an x-­y-­coordinate system. In each lasers is tuned to match the molecular vibration of interest, that
point, only a small dimension (typically below 1 μm) is illuminated is a specific vibration of a drug substance or excipient. By creat-
with the laser and a full spectrum from each point is recorded. ing a coherent, stimulated condition, signals many orders higher
Subsequently, a data cube is generated comprising the spectra than in conventional Raman spectroscopy are created. 22,23 In
and x-­y-­coordinates. From this datacube, parts of the information CARS, the molecular vibration, the lasers have been tuned to,
are extracted to form, for example a 2D colour-­coded image of creates a photon in the anti-­Stokes part of the spectrum which
a drug substance inside a skin sample. This part of information is then detected. 22-­24 In SRS, the energy transfer from the Stokes
1314 | LUNTER et al.

to the pump laser is detected via a modulation transfer scheme.

The CARS spectrum may be contaminated by non-­resonant con-
tribution which makes quantification challenging. Further, signal-­
concentration dependence is not linear but quadratic which needs
to be compensated for using appropriate algorithms. 22,23,25,26 The
SRS spectrum does not contain non-­resonant contamination and
the signal-­concentration dependence is linear. 27 Both methods
are most powerful when a specific molecular vibration of the sub-
stance under investigation either falls within the range of 2000–­
2700 cm−1, where there are no vibrations of the skin itself, or if
F I G U R E 1 Left: schematic of the Rayleigh criterion, right:
they are deuterium labelled which makes vibrations fall into this
24,25,26,28,29,30 illustration of depth-­dependent attenuation and laser focal volume
range, too. In this range, the methods show limits of
broadening inside a sample
detection around 100 mM but this may increase drastically when
signals overlapping with those of the skin are to be used. 23 Tuning
the lasers to multiple frequencies, that is acquiring hyperspectral SERS probes will probably lead to more versatile instrumentation
data would improve this situation as more than one wavenumber in the future making the method more convenient and increase its
could be used for evaluation but this drastically slows down ac- use in skin research. 33,35,37
quisition speed. Further, many setups have limited tuning range. 31

4.5 | Confocal Raman spectroscopy/microscopy

4.4 | Surface-­enhanced Raman scattering (SERS)
CRS is by far the most frequently used Raman technique to investi-
Raman spectroscopy of the skin is often hampered by fluorescent gate penetration of exogenous substances into the skin or to analyse
background in the Raman spectrum and superposition of the sig- the skin in healthy or diseased state. Combined with a movable scan
nals of interest with the spectrum of the skin itself, making it dif- stage that enable precise positioning of the sample in three dimen-
ficult to generate high signal-­to-­n oise ratios. Surface-­e nhanced sions, imaging in two or three dimensions can be performed, also
Raman spectroscopy (SERS) uses nanostructured metal surfaces referred to as confocal Raman microscopy. A confocal Raman micro-
to generate Raman spectra that are several orders of magnitude scope consists of a laser light source, objective(s), beam splitter, pin-
higher than regular ones. To this end, a very thin sample speci- hole, motorized scan stage, detector and optical fibres to guide the
men is adsorbed to a nanostructured metal surface like gold or light from the laser source to the objective and from the objective
silver. 32,33 When illuminating the sample with an excitation wave- to the detector. Furthermore, the fibres may also be used as pinhole
length that matches the plasmon resonance of the atoms at the to achieve confocality. The principle of confocality and the factors
surface of the specimen, a strong electromagnetic field is induced. affecting it as wells as resolution will be explained briefly hereafter.
The generated electromagnetic wave propagates along the sur- As in every optical method, the resolution is defined by the
face of the specimen. As the Raman signal intensity relates to Rayleigh criterion (Equation 1).38
the amplitude of the electromagnetic field, the Raman modes of
Δx = 0.61∗ 𝜆 ∕ NA (1)
the molecules close to the metal surface are strongly enhanced.
As a result, the incident and the scattered lights intensities are
increased. 33,34 Raman signal intensity enhancement can be ex- where Δx is the distance or resolution or laser focal volume [nm], λ is
plained by the high intensity of the electromagnetic field near the wavelength of the light [nm] and NA is the numerical aperture of
the plasmonic substrate and thus by classical electromagnetic the objective.
theory. On the contrary, there are also chemical enhancement Figure 1 shows an illustration of the Rayleigh criterion. On the
effects. They are derived from the interactions of the samples' left, the distance x between two point-­like light sources is larger
molecules with the SERS substrate and can be explained by quan- Δx so that the two points can be differentiated. The situation in
tum mechanical electronic structure theory. 33 SERS requires the the middle shows two point-­like structures at a distance x which is
sample to be prepared as a very thin sheet, carefully fixed to the smaller than Δx. The two points cannot be distinguished and ap-
metal surface which makes sample preparation rather elaborate. pear as one spot. The distance Δx beyond which two point-­like light
SERS substrates themselves require multiple factors to be care- sources cannot be detected as two points depend on the wavelength
fully balanced which makes them elaborate to prepare as well. of the light and the numerical aperture of the objective. The longer
Furthermore, depth profiling is not feasible unless a large number the wavelength of the light and the smaller the numerical aperture
of such very thin samples are prepared. The technique is therefore of the objective the larger Δx will become. As a result, resolution
only rarely used in skin penetration analysis. 32,35,36 Technical pro- decreases. The used wavelength is determined by the laser used
gress such as tip-­e nhanced SERS or the fabrication of microneedle and can only be altered if different lasers are at hand. The numerical
LUNTER et al. | 1315

aperture is defined by the objective used and can more easily be pinhole is installed between the objective and the detector. The
altered by simply attaching different objectives to the microscope. principal setup is shown in Figure 2-­right. Only light from the focal
In general, water or oil immersion objectives show higher numer- plane will be allowed to pass through the pinhole and will be guided
ical apertures compared to metallurgical objectives.39-­42 Typically, on towards the detector. Light from different depths, for example
green (532 nm) or read (785 nm) lasers are used in confocal Raman the reference plane will not be able to pass the pinhole. The size of
spectroscopy. With these lasers and an oil immersion objective with the pinhole determines the height of the focal plane and thus deter-
the numerical aperture of 1.25, the maximum theoretical resolution mines the degree of confocality and depth resolution.39-­42 A small
which can be achieved is 260 nm or 383 nm, respectively. In practice, pinhole and high degree of confocality are of pivotal importance in
the resolution also depends on the quality of the objective as well skin depth profiling in order to reject all rays from out of focus re-
as on the sample and the interaction of the light with the sample. In gions. Otherwise, spectra of substances, for example the drug on
depth profiling, a laser is focused to different depths inside the skin the skin surface may be erroneously detected inside a certain skin
and spectra are acquired from these depths. Inside the sample, the depth and lead to false penetration profiles.43,44
light is scattered and absorbed which leads to depth-­dependent sig- In principle to types of microscope setups exist, upright and in-
nal attenuation. Furthermore, the laser focal volume broadens and verted microscopes. For skin Raman analysis, upright microscopes
the resolution therefore decreases with increasing depth inside the are used for in vitro or ex vivo studies whereas inverted microscopes
sample (Figure 1, right). As a result, spectra can only be obtained are used for in vitro cell culture and in vivo measurements. In the
from up to 50 μm inside the skin. To counteract the effect of depth upright setup, the skin sample is placed onto a movable scan stage
attenuation, the spectra acquired from deeper depths inside the skin and beneath the objective.43,44,46,47,48,49 The distance between ob-
need to be normalized to the signal at the skin surface. Otherwise, jective and sample can be adjusted by the scan stage. Thereby, the
the decrease in drug concentration with increasing depth in the skin laser will be focused into different depths of the skin. In most cases,
will be overestimated.43,44 the skin is incubated beforehand in Franz diffusion cells or transwell
When light is focused into a sample of different refractive index, plates. The samples are then withdrawn from the cells or plates and
the incident light suffers from refraction at the sample surface. As mounted onto special sample holders which guarantee skin hydra-
shown in Figure 2 left, the laser light will be focused into deeper tion throughout the Raman analysis.43,44,50,51 A more recent devel-
depth of the sample if the laser is directed from a medium with the opment is a modified diffusion cell which can be mounted directly
lower refractive index into a medium with higher refractive index beneath the objectives of the microscope. The setup (Figure 3B)
(the sample). Although the light is theoretically focused into the enables the incubation of the skin with the respective solution of
depth Δ, it will be refracted and focused into the depth z. To avoid formulation directly underneath the Raman microscopes objective.
this effect, an immersion medium with the same refractive index as Thereby, Raman spectra can be acquired at any timepoint, enabling
39-­4 4
the sample should be used as shown in Figure 2 middle. In this in situ analysis of penetration kinetics.46,52
case, no refraction takes place at the interface between immersion For in vivo analysis, an inverted microscope setup is used. It com-
medium and sample and the depth z the laser is actually focused to prises an oil immersion objective which is located beneath a measur-
equals the theoretical depth Δ. In the case of skin, oil immersion ob- ing window onto which the respective body part may be placed for
jectives are helpful as the refractive index of the immersion oil (1.51) analysis. The depth into which the laser is focused is adjusted through
is close to that of the stratum corneum (SC) (1.47).45 the distance between objective and measuring window.53-­55 The use
The light which is focused onto or into the sample by the ob- of the inverted setup shows the advantage that the skin is always in
jective of the microscope interacts with the sample and is scat- full contact with the measuring window and will thus not be moved out
tered. The scattered light is then collected by the same objective of focus due to involuntary movements of subjects. A disadvantage is
and guided towards the detector. To achieve confocality, an optical that (at least to date) this system does not allow for measurements in

F I G U R E 2 Illustration of reflection—­sample surface, and microscope setup of confocal microscopy. Left: metallurgical objective, middle:
oil immersion objective and right: the propagation of light through a confocal microscope
1316 | LUNTER et al.

F I G U R E 3 The schematic profiles of the off-­line device (A) of Franz diffusion cell for skin incubation and in-­line device (B) of skin
incubation cells used (reproduced from Pharmaceutics 2021, 13, 67. https://doi.org/10.3390/pharm​aceut​ics13​010067; Creative Commons
Attribution (CC BY) licence (https://creat​iveco​mmons.org/licen​ses/by/4.0/)) 46

F I G U R E 4 Number of publications in PubMed database with the search term (A) “Raman spectroscopy” and (B) “Raman spectroscopy
skin.” Changes over time (C) in the proportion of skin research in all Raman spectroscopy articles and (D) in the number of publications on
Raman spectroscopy in skin research regarding in vivo/ in vitro/ ex vivo applications (The PubMed search was finalized by 25 February
2022)
LUNTER et al. | 1317

two or three dimensions thereby prohibiting imaging/mapping. Solving task such as Attenuated Total Reflection Fourier Transform Infrared
this issue should only be a matter of software capabilities, and it can be Spectroscopy, CRS has been shown to provide significantly richer
expected that this drawback will be resolved in the future. spectroscopic detail and to differentiate between SC and underly-
ing epithelial layers in skin tissue sections of human skin and artifi-
cial skin models.65 Data on skin biochemistry by CRS has stepwise
5 | R A M A N S PEC TROS CO PY I N S K I N been established for intact and diseased skin in the last 20 years. The
R E S E A RC H FRO M TH E B EG I N N I N G S BA S E D steps along the way are discussed in the following section and recent
O N PU B M E D DATA BA S E examples are given.
CRS has been used to study the molecular composition of human
Though the Raman effect underlying the Raman spectroscopy was de- skin in vivo,66,67 tissue sections ex vivo65,68,69 and skin substitutes
scribed in 1928, it could become widespread only after the invention in vitro.70-­72 The Raman spectrum of human SC is dominated by the
of lasers in the 1960s. The PubMed database contains 15 publications vibrational bands of its structural proteins, amino acids and lipids.
between 1947 and 1960 with the keywords “Raman spectroscopy.” In Both human skin and animal skin can be analysed by CRS (Figure 5).
the next decade, an average of less than 5 publications per year was Spectral bands were first assigned by Barry et al.73 and are sum-
published, followed by a few dozen articles per year in the 1970s, when marized in recent literature.35,74 The most prominent bands are
the microscopic Raman with an optical microscope equipped with a given in Table 1.
Raman spectrometer has also been invented. Since the introduction of Changes in barrier function of a reconstructed human epidermis
the near-­infrared region Fourier transform Raman spectroscopy ena- model caused by frozen storage have been studied.75 Both storage
bled the application of the technique in biomedical studies, the number time and temperature were relevant factors. Molecular properties of
of relevant papers has steadily increased, reaching more than 1000 human skin in vivo have been analysed for different age groups; dif-
articles in 2005 and over 4400 in 2021, as it is shown in Figure 4A,B. ferences in skin biochemistry have been shown for elderly76,77 and
There are a few articles between 1975 and 1990 which are re- infant skin.78
lated to skin studies, but in these cases, they are mostly about the Apart from human skin, animal skin models have been assessed.
study of different molecules isolated from the skin (e.g. peptides Porcine skin is perhaps most frequently used due to its relative
isolated from the skin of Xenopus laevis, 56 or proteodermatan sul- similarity to human skin in morphology and barrier function.79-­81
phate from pig skin57). The first studies on the skin composition were Differences of skin models to their natural counterpart based on
published in the late 1990s (e.g. comparing the structure of water, CRS analysis of their molecular composition provide insights into
proteins and lipids in intact human skin,58 or analysing the molecular the underlying cause of lower barrier function as known for recon-
structure of 5200-­year-­old skin59). The annual number of papers on structed skin models and porcine skin ex vivo. Choe et al.79 con-
the use of Raman spectroscopy in skin research has already reached ducted an in-­depth analysis of SC molecular profiles of human skin
100 in recent years. Which means that, although this is only a few in vivo and porcine ear skin. The SC depth profiles of porcine skin
per cent (2%–­3%) of all Raman spectroscopy papers, its proportion exhibited lower natural moisturizing facture (NMF) content through-
is slowly increasing, presented in Figure 4C,D. out the entire SC and depth-­dependent differences in hydrogen
Nearly one third of hits on Raman spectroscopy in skin research bonding states of water. A higher content of keratin in β-­sheet form
are not labelled either in vitro, in vivo or ex vivo, including, for ex- and higher hexagonal lateral packing order of intracellular lipids was
ample, structural studies of molecules isolated from skin. A further observed at 10%–­50% SC depth, which permits penetration of both
one third of the articles deals with in vivo applications, such as diag- hydrophilic and lipophilic entities more easily than in human skin.
60,61 62
nosis of skin cancer, or monitoring the changes due to ageing. Of particular interest when investigating the effect of dermal
The remaining part of the relevant papers used ex vivo analysis for preparations is skin lipid composition.53,82 The effect of surfactants
instance for cancer discrimination,63 or for classification of burn on the SC lipid matrix can be observed by CRS through changes in
severities,64 and in vitro applications, such as using in vitro skin lipid content via lipid/protein ratio, changes in conformation and
substituents. in lateral packing order.83,84 Non-­ionic polysorbate emulsifiers and
ethoxylated ethers with a small number of oxyethylene groups were
found to cause smaller lipid perturbations than, for example PEG-­
6 | U TI LIZ ATI O N O F R A M A N 20 ethers.84 Further, the NMF as a collection of hygroscopic mol-
S PEC TROS CO PY I N S K I N R E S E A RC H ecules derived from the protein filaggrin in the SC can be analysed
by CRS; the contribution of free amino acids such as serine, glycine,
6.1 | Skin composition studies pyrrolidone-­5-­carboxylic acid, arginine, ornithine, citrulline, alanine,
histidine and urocanic acid is usually taken into account to obtain
Confocal Raman Spectroscopy (CRS) is being extensively used to information on the state of skin hydration and barrier function.82
analyse skin composition. The basic analysis of skin compounds The analysis of NMF in atopic skin has been validated against tape
under physiological conditions comprises a large body of data. stripping/HPLC analysis; comparable results were obtained with
Among other vibrational spectroscopic techniques available for this both methods.85
1318 | LUNTER et al.

F I G U R E 5 Model Raman spectra of fingerprint region (left) and high wavenumber region (right) of healthy human skin in vivo (A, B) and
porcine ear skin ex vivo (C, D). Images were taken with a gen2-­SCA Raman spectroscope (River D., the Netherlands)

The water content within the skin, in particular the SC, is im- within the SC, however, was associated with disordered lipid and
portant to keep the skin barrier functional and is therefore routinely protein states. Recently, the application of heavy water was investi-
investigated in dermatological studies. CRS can be used to calculate gated by CRS89; different ethoxylated emulsifiers were dissolved in
the water mass concentration profile as a function of depth based D2O to probe their effect on skin biochemistry while avoiding addi-
on the ratio between the intensity of the OH vibration and keratin tional effects on the OH bond of the skin spectrum through exter-
vibration.82 Skin hydration, that is the water molecules present in nally applied water. The D2O distribution within the skin was well
the SC that are involved in maintaining elasticity and physiological discernible due to its OD stretching band and was clearly affected
77,86,87
molecular structures, has been intensively studied by CRS. by the co-­applied selected ethoxylated emulsifiers in dependence
For the mentioned aspects, the bound water content is of particular of their chemical structure and the positive control sodium laureth
importance since it is associated with intracellular lipids and proteins sulphate (SLES).
via hydrogen bonds.86 In this context, the hydrogen bonding state The effect of dermally applied preparations on hydration status
of water as determined by CRS was found to reach a maximum at and NMF levels has been explored by CRS for washing procedures
a SC depth of 30% of its entire thickness, correlating well with the with different cleaning products90,91 and cosmetic emollients,92 but
maximum lateral packing order of the intercellular lipid matrix and also for drug delivery systems in vivo.93,94 Normal skin, but also the
86
the NMF content. Atmospheric relative humidity was reported lip region have been investigated.95 Titanium dioxide nanoparticles,
to affect the partially bound states of water within the SC.88 At as frequently used in cosmetics for sun protection, were found to
intermediate relative humidity around 60% both lipid organization promote structural rearrangement of the SC lipid bilayers under
and protein deployment were optimal, representing the maximal controlled indoor illumination,96 which underlines the advantages of
SC water binding capacity. An increased content of unbound water CRS in toxicological investigations.
LUNTER et al. | 1319

TA B L E 1 Assignment of spectral bands

Raman shift Raman shift
in healthy human skin after35
[cm− 1] Assignment [cm− 1] Assignment

526 ν (SS) 1274 ν (CN), δ (NH) amide 3 α-­helix

600 ρ (H) 1296 δ (CH2)
623 ν (CS) 1385 δ (CH3) symmetric
644 ν (CS) amide 4 1421 δ (CH3)
746 ρ (CH2) in phase 1438 δ (CH2) scissoring
827 δ (CCH) aliphatic 1552 δ (NH), ν (CN) amide 2
850 δ (CCH) aromatic 1585 ν (C=C) alkenic
883 δ (CH2), ν (CC), ν (CN) 1652 ν (C=O) amide 1 α-­helix
931 ρ (CH3) terminal, ν 1743 ν (C=O) amide 1 lipid
(CC) α-­helix
956 ρ (CH3), δ (CCH) 1768 ν (COO)
alkenic
1002 ν (CC) aromatic ring 2723 ν (CH) aliphatic
1031 ν (CC) skeletal cis 2852 ν (CH2) symmetric
1062 ν (CC) skeletal trans 2883 ν (CH2) asymmetric
1082 ν (CC) skeletal random 2931 ν (CH3) symmetric
1126 ν (CC) skeletal trans 2958 ν (CH3) asymmetric
1155 ν (CC), δ (COH) 3050 ν (CH) alkenic
1172 ν (CC) 3280 ν (OH) of H2O
1244 δ (CH2) wagging,
ν (CN)amide 3
disordered

Note: ν, stretch; ρ, rock; δ, deformation.

Apart from the effect of chemical stressors, the impact of glycation end products as sign of photo-­damage and skin ageing
physical procedures such as tape stripping, microneedling or were successfully analysed in human biopsies by CRS.102
hair removal, is possible, but less promising since not all physical
stressors evoke significant changes in the chemical composition
of the skin. In addition, potential changes in SC thickness due to 6.2 | Skin penetration studies
physical removal of corneocytes have to be taken into account in
data evaluation.97,98 In a recent study, the effect of iontophoresis In analogy to its role in skin composition analysis, CRS has found
and sonophoresis on barrier function alone and in combination a solid place in skin penetration studies. In comparison with other
with a chemical enhancer was shown for a model permeant using vibrational spectroscopic techniques used to analyse drug pen-
e nhanced Raman spectroscopy 99; the
mouse skin and surface-­ etration, for example the ATR-­
F TIR-­
spectroscopy/tape stripping
best option for oxaprozin delivery was found to be sonophoresis method, it is non-­
invasive, faster and requires less sample pro-
combined with azone as enhancer. cessing.54 Thus, CRS has become an important asset in cosmetic
Chemical and physical influences held aside; skin is also affected and pharmaceutical formulation development. Likewise, it is used
by photo-­stress, such as solar radiation. Studies with artificial skin to evaluate potentially hazardous permeants in toxicological stud-
sections subjected to simulated solar radiation cell culture studies ies. For these tasks, different membranes are used: human skin in
have shown that induced biochemical changes, for example in SC vivo,103 human skin ex vivo,104,105 in vitro skin substitutes75 and por-
lipid profile can be detected significantly earlier by CRS than by con- cine skin.52,54,106,107,108,109 Rodent skin110 is rarely due to its inherent
100
ventional cytotoxicological analyses. Despite biochemical differ- differences to human skin.
ences of the probed model to actual human skin, this approach using Caffeine,52,106,109,111,112,113 lidocaine,104 procaine,107,114 tetra-
multivariate data analyses is an interesting prospect for diagnosis of caine,115 salicylic acid,113 ibuprofen,103 flufenamic acid,105 imiqui-
subclinical skin damage due to the high sensitivity of CRS. Recent mod,116 trans-­retinol,117,118 resorcinol,75 niacinamide,119 sulfathiazole
studies confirmed similar effects caused by infrared irradiation in sodium,54 triamcinolone acetonide120 and oxaprozin are among the
human skin in vivo,101 including increased SC lipid content and de- most investigated permeants in the CRS penetration studies.99 Next
creased basal deoxyribonucleic acid (DNA) peaks. The observed to these drugs, the penetration of enhancers such as DMSO,54,121
spectral shift of the amide I peak of collagen in the dermis indicates propylene glycol,103,106,107,118 polyoxyethylene-­23-­lauryl ether107
structural changes and functional loss. In a recent study, advanced and other ethoxylated emulsifier systems114,122 has been evaluated.
1320 | LUNTER et al.

The skin penetration of classic anionic surfactants such as sodium 6.3 | Skin diseases
dodecyl sulphate (SDS) or sSLES has likewise been investigated in
numerous studies, often as positive control.54,123,124 Regarding skin As mentioned before, articles on the applicability and utility of
penetration of additives used in cosmetic or pharmaceutical prepa- Raman spectroscopy for dermatological purposes first appeared in
rations, data on the penetration behaviour of oils or waxes,95 preser- the 1990s.1,2 Shortly afterwards, increasing number of results were
vatives120 or sunscreen agents125 have been presented. published on Raman spectroscopic investigation of cancerous skin
In dependence of the experimental setup used, both two-­and tissues.
three-­dimensional information on the location of applied permeants Next to healthy skin, analysis of pathological conditions has been
is obtained. To facilitate visualization of drugs next to solvents or the target of numerous CRS investigations, for example on atopic
enhancers such as octanol or propylene glycol and to obtain lateral dermatitis128,129 and psoriatic lesions.130 A very specific application
spatial distribution of the applied materials, the use of deutered in context with unphysiological skin conditions is the identification
compounds is a valid approach.105 When using complex vehicles, of multicoloured pigments in tattooed human skin preceding laser
overlapping of formulation compounds with numerous skin bands removal.131 The effect of applied substances on skin biochemistry,
has to be avoided.104 However, monitoring the spatial distribution in particular on the SC lipid matrix and barrier function, has been
of chemically similar species in CRS penetration studies, for example investigated for numerous common additives in dermal prepara-
prodrug and drug, is possible without labelling for compounds with a tions: oils,95,132 emulsifiers,54,83,84,133,134 solvents or other penetra-
125
suitable spectroscopic profile. tion enhancers.52 Strategies have been proposed on how to optimize
The experimental conditions for CRS penetration studies should biochemical analysis of the skin by CRS considering spectral variabil-
be kept constant during the analysis. Artefacts can arise from ity,69 impact of applied chemicals87 or keratin distribution through-
changes in skin biochemistry during analysis caused by prolonged out the SC.135
measurement time (occlusion, changes in temperature, etc.). Skin Skin cancer is one of the common cancers. Several types of
temperature and hydration of porcine skin samples were found to melanomal and non-­melanomatous (basal cell carcinoma (BCC) and
affect results,109 potentially due to crystallization of the applied squamous cell carcinoma (SCC)) cancers136 can be distinguished. The
drug. This should be kept in mind when comparing results obtained first step in the medical diagnosis of the disease is a visual exam-
with different experimental setups. ination,137 followed by skin biopsy and histopathology.136,137 Thanks
An important question in CRS penetration studies is how to ob- to increasingly sophisticated data processing methods, it became
tain quantitative drug penetration profiles. So far, most studies have possible to fully differentiate between cancerous and non-­tumorous
delivered information on permeant distribution within the skin on tissues3-­5 and between different types of skin cancers, and to de-
a relative scale. Approaches for quantitative analysis of permeants termine a demarcation line of the diseased tissue.6-­9 The technical
have been proposed.126 In recent years, Caspers et al.119 have moved advances have made it possible to apply the technique clinically for
forward in this respect; the first fully quantitative approach analys- examination and diagnosis of various, primary skin cancers.10-­15 On
ing the skin penetration of niacinamid from various formulations application of Raman spectroscopy for diagnostic purposes, some
into human skin was studied both in vitro using classic Franz-­t ype sample papers are shown in Table 2.
diffusion cells and in vivo by quantitative CRS. The correlation
after linear regression between cumulative drug amounts in vitro
and penetrated drug at 2 μm SC depth in vivo was reported with 6.4 | Applications in cosmetoscience
R 2 = 0.98. Further research will provide more insight into quantita-
tive CRS analysis for different applications to determine its role in In addition to the dermatological use of Raman spectroscopy, its use
future bioequivalence studies. for cosmetology purposes is gaining ground, as it is becoming im-
Among the most important conclusions derived from the sum- portant to know the mechanism, exact extent and depth of penetra-
marized penetration data is the importance of selecting suitable ex- tion of different active ingredients and drugs furthermore provides
perimental parameters for CRS instrumentation such as choice of information on the distribution of these molecules in different layers
objective and pinholes,43,44 and skin samples. Recently, penetration of the skin. In vivo Raman spectroscopy is a non-­invasive, sensitive
studies performed in parallel with a common multi-­purpose confocal method that offers an effective, easy-­to-­use solution for all these
Raman spectroscope by WiTec (alpha 500) and the River Diagnostics requirements.
127
device (gen2 SCA) were found to deliver comparable outcomes. The penetration of several popular molecules in the cosmetic
Binder et al. obtained similar enhancement ratios for procaine HCl industry has been investigated by Raman spectroscopy. The tested
when using polyoxyethylene23-­lauryl ether to promote skin pene- active ingredients provide solutions to various skin problems (e.g.
tration. This is promising considering that instruments of two dif- acne, skin ageing, hydration, anti-­inflammatory problems and sun
ferent suppliers were used in different laboratories, working with protection). Essendoubi et al. studied three different hyaluronic acid
different objectives, pinholes and laser wavelengths. Aspects to be derivatives (Cristalhyal (1000–­1400 kDa), Bashyal (100–­300 kDa)
kept in mind are the specific mode of data evaluation and substance-­ and Renovhyal (20–­50 kDa)) on plastic abdominal skin samples re-
specific aspects such as resonant effects.127 moved by plastic surgery.138 Tfaili et al.139 performed penetration
LUNTER et al. | 1321

TA B L E 2 Examples for diagnostic application of Raman spectroscopy in dermatology (modified from Franzen et al. and Zhao et al.35,160)

Type of investigation Type of the skin Result, conclusion Ref.

In vitro Human SC samples Comparison of Raman spectroscopy and infrared spectroscopy 73,161
based on spectra taken from SC, supporting the
dermatological applicability and utility of Raman spectroscopy
In vitro Biopsies Differentiation of basal cell carcinoma (BCC) from non-­tumorous 162-­164
BCC tissue based on Raman spectra
In vitro Biopsies healthy and cancerous Discrimination of diseased and non-­c ancerous tissues, 165
skins differentiation of skin cancer types
In vitro Biopsies and normal skin Investigation the differences between the Raman spectra of 166-­169
samples malignant melanoma (MM), basal cell carcinoma (BCC),
pigmented naevi (PN), seborrheic keratoses (SK) and normal
skin for diagnostic purposes
In vitro Healthy SC samples, atopic Analysis of skin samples from patients with atopic dermatitis and 130
dermatitis, psoriasis psoriasis by Raman spectroscopy
Ex vivo SC isolated from human Investigation and understanding of the hydration process 88
abdominal skin
In vivo Psoriatic skin Examination of psoriatic skin by Raman spectroscopy 170
In vivo Allergic skin Application of Raman spectroscopy in patients suffering from 171
nickel allergy
In vivo Atopic dermatitis Use of Raman spectroscopy in patients suffering from atopic 172
dermatitis
In vivo Skin cancer Application of Raman spectroscopy for in vivo cancer diagnosis 137

studies with caffeine, also on human abdominal skin samples. The authors reported that no statistically significant effects on any of
penetration of cholesterol-­and linoleic-­containing creams has been the observed parameters were obtained, indicating good skin toler-
studied in the skin of healthy volunteers. It was determined that the ability of all tested formulations.
active ingredients could penetrate into the stratum corneum and In addition to penetration studies, Raman spectroscopy is also
have a repairing and regenerating effect during their release from used in formulation studies (e.g. for glycolic acid, Punica granatum
the topical cream formulation.140 Dancik75 examined the effect of seed oil hydroxyphenethyl ester [PHE] and raspberry seed oil).144-­147
freezing temperature and duration on the resorcinol penetration Effects of glycolic acid on the skin were assessed by measurement
on reconstructed human epidermis (RHE) samples. To evaluate the of biophysical skin parameters in in vivo studies (5-­h, 4-­h and 7-­day).
safety and the synergistic effects of tea tree, lavender, eucalyptus Organic pigments are favoured for tattooing because of their
and tangerine essential oils in combination on the skin141 Infante and high tinting strength, light fastness, enzymatic resistance, disper-
co-­workers (2022) used ex vivo confocal Raman spectroscopy and sion and relatively inexpensive production costs. A methodology
in in vitro and in vivo conditions. Skin penetration was evaluated was established by Poon and co-­workers (2008)148 using micro-­
after direct application of essential oils to pig ears. The authors con- Raman spectroscopy on an animal model to correctly identify the
cluded that the essential oils in combination were safe and effective constituents of a selection of modern, organic tattoo inks in situ or
in the improvement of the hydrolipidic balance and morphological postprocedure, within the skin. The results reported by Persechino
properties of the skin. Micek and co-­workers (2021)142 investigated et al (2019) and Carlson et al (2016) confirm the high sensitivity and
the effects of two creams: 3% S. balsamita extract and 3% Taxifolin great expendability of Raman spectroscopy for identification of
(TXF) on the function of adult skin. The percutaneous penetration pigments present in tattoo inks and the relative quality control (as
of creams was also examined with the use of electrospray ioniza- composition control).149,150 The aim of this research was to assess
tion mass spectrometry (ESI-­MS) and confocal Raman spectroscopy. the feasibility of Raman spectroscopy as a screening technique for
The 3% S. balsamita extract cream reduced hyperpigmentation, er- chemical characterization of tattoo pigments in pathologic reacting
ythema and elevated pH. A higher penetration rate was revealed for tattoos and tattoo ink products to depict unsafe pigments and me-
the 3% TXF cream than for the 3% S. balsamita extract cream by tabolites of pigments.
Raman spectroscopy. Based on the results, the authors concluded
that both extracts can be considered as ingredients of skincare
products for adults. Pany and co-­workers (2021)143 tested the effect 7 | A RTI FI C I A L I NTE LLI G E N C E I N
of hydroxypropyl-­β-­cyclodextrin (HP-­β-­CD) in cosmetic submicron A N A LYS I S O F R A M A N S PEC TR A
emulsions and submicron emulsion gels on physiological skin param-
eters during regular application in a clinical setup. Confocal Raman The large amount of data from Raman spectroscopy allows, and at the
spectroscopy was employed to monitor urea and NMF levels. The same time may require the use of artificial intelligence in data analysis
1322 | LUNTER et al.

to extract meaningful information and make predictions based on 8 | R EG U L ATO RY A S PEC T S O F R A M A N

the data collected. In the literature, a myriad of different application S PEC TROS CO PY I N S K I N TE S TI N G
fields is available, where the measured Raman spectra were the input
data of machine learning, mostly by classification algorithms. A large Modelling drug permeation through the skin is a complex challenge.
portion of the studies focuses on non-­medical applications, with an Although there are many quantitative and qualitative methods for
emphasis on adulteration detection in the food industry. Other part following-­up skin penetration, the different techniques are not fully
of the articles describe Raman spectroscopic techniques combined equivalent but complement each other. Therefore, the regulation of
with machine learning algorithms to improve diagnostic measures dermal and transdermal formulations has received increasing atten-
for identifying diseases such as infections, cancer, neurodegenera- tion nowadays. There are more and more guidelines to provide har-
tive disorder or different skin conditions. A summary on application monization for dermal and transdermal testing. The Organization for
of artificial intelligence in Raman spectroscopic data analysis can be Economic Cooperation and Development (OECD) published several
found in Table 3. documents about this topic, including: Guidance Notes on Dermal

TA B L E 3 Summary of recent studies combining Raman spectroscopy with machine learning algorithms

Machine Learning
Sample Details methods Ref.

Non-­medical Butter Detection of adulteration of butter with margarine PCA, PCR, PLS, ANN 173
applications Caviar Discrimination between three different caviar types PCA, LDA, ANN 174
Edible oils Edible oil authentication (sesame, hemp, walnut, Ensemble classifier-­ 175
linseed, pumpkin, sea buckthorn) subspace KNN when
the PCA was disabled
Fruit distillates Trademark fingerprint differentiation, geographical DT, DA, SVM, KNN 176
discrimination other ensemble classifiers
Honey Detection of low-­concentration adulterated Suichang CNN, PNN, SVM 177
native honey
Milk Differentiation between milk from different species PCA, RF 169
(cow, buffalo, goat and human)
Minerals Recognition of minerals and estimation their elemental CNN, KNN, SVM, 178
composition extremely
randomized trees,
weighted-­neighbours
Poplar Prediction of the lignin content DT, SVM ensemble 179
in poplar wood samples classifiers (LightGBM,
CatBoost, XGBoost)
Medical Alzheimer's disease Alzheimer's disease diagnosis based on the analysis of ANN, SVM-­DA 180
applications cerebrospinal fluid
Alzheimer's disease Alzheimer's disease diagnosis based on the analysis of ANN, GA 181
saliva
COVID-­19 infection Diagnosis of COVID-­19 infection based on saliva MIL 182
samples
Tuberculosis infection Distinction between tuberculosis positive (diseased), PCA, HCA 183
negative (cured) and control (healthy) serum
samples
Breast cancer Classification of breast cancer subtypes PCA-­DFA, PCA-­SVM 184
Colorectal cancer Prediction the effect of immunotherapy SVM, RF 185
Lung cancer Cytopathological diagnosis of lung cancer KNN, SVM 186
Skin cancer (basal/ Distinction between basal cell carcinoma, squamous CNN, LR, SVM 187
squamous cell cell carcinoma and healthy skin tissues and cells
carcinoma)
Skin cancer (melanoma) Distinction between benign versus malignant LightGBM, KNN, XGBoost 188
melanoma tissues
Atopic dermatitis Stratification of severity in atopic dermatitis SVM 189
Burn injury Classification of burn injury LR, SVM, RF 190
LUNTER et al. | 1323

Absorption (No. 156),151 Test Guideline 427 (in vivo methods)152 Spectroscopy at University of Tours and also to Hichem Kichou for
153
Test Guideline 428 (in vitro methods), and Guidance Document the excellent practical training in skin analysis.
for the Conduct of Skin Absorption Studies.154 There are some other
documents: World Health Organization International Programme on AU T H O R C O N T R I B U T I O N S
Chemical Safety (WHO/IPCS) Environmental Health Criteria 235,155 DL: Raman spectroscopic techniques, VK: Utilization of Raman
European Centre for Ecotoxicology and Toxicology of Chemicals spectroscopy in skin research, DK: Analysis of PubMed database for
(ECETOC) Monograph 20,156 United States Environmental Protection Raman spectroscopy; artificial intelligence for evaluation of Raman
Agency (USEPA) report on dermal exposure assessment,157 European results, FE, ZV-­M: Applications of Raman spectroscopy in skin dis-
Food Safety Agency (EFSA) Guidance on dermal absorption for plant eases and cosmeto-­science; ZV-­M: list of references SB: Principle of
protection products,158 and the European Medicines Agency (EMA) Raman spectroscopy and regulatory aspects, FE: History of Raman
document, Draft Guideline on Quality and Equivalence of Topical spectroscopy, introduction and summary.
Products.159 There are two methods proposed in these guides: the
widely used diffusion cell and tape stripping methods. The Raman F U N D I N G I N FO R M AT I O N
spectroscopy is mentioned only in the latest EMA recommendation, This work was supported by Le STUDIUM “Smart Loire valley”
so it is very important to investigate the method's applicability, and program, 2022, France and by National Research, Development
in the future, if there will be a sufficiently favourable investigation, it and Innovation Office of Hungary through the grant
might be recommended by all authorities. TKP2021-­​EGA- ­42.

C O N FL I C T O F I N T E R E S T
9 | S U M M A RY No conflict of interest.

Simplicity in using and collecting data, as well as conducting analyses DATA AVA I L A B I L I T Y S TAT E M E N T
without the need for prior labelling and complicated sample prepara- Data sharing not applicable to this article as no datasets were gener-
tion, has resulted in increased interest and a significant increase in the ated or analysed during the current study.
use of Raman spectroscopy in the field of life sciences including skin
research and dermatologic diagnosis. Raman spectroscopy techniques ORCID
used for research on biological material, that is skin tissues, provide Victoria Klang https://orcid.org/0000-0003-2561-4378
insight into the structure and organization of the dermal barrier. Raman Dorottya Kocsis https://orcid.org/0000-0001-7908-3248
microscopy is a precise tool for the study of the structural polymers, Franciska Erdő https://orcid.org/0000-0001-6265-3777
metabolites, lipids, proteins and water content in the tissues. Owing
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