Fluorescence spectroscopy of skin$

Nikiforos Kolliasa,*, George Zoniosb, Georgios N. Stamatasa

aJohnson and Johnson Consumer Products Worldwide, 199 Grandview Road, Skillman, NJ 08558, USAbWellman Laboratories of Photomedicine, Massachusetts General Hospital, Boston, MA 02114, USA

Received 13 October 2000; received in revised form 20 June 2001; accepted 20 June 2001

Abstract

The fluorescence properties of skin both human and of an animal model are reviewed in this article. Fluorescence

spectroscopy is a valuable tool for the investigation of the optical properties of the skin in the ultraviolet range of the spectrum

because the absorption bands are confluent. Epidermal and dermal fluorescence signals are each produced in distinct spectral

regions. Biological processes modulate the fluorescence signals in predictable ways. Such cases include aging, epidermal

proliferation, and photoaging as well as diseases such as psoriasis, acne and non-melanoma skin cancer. Fluorescence excitation

spectroscopy has proved a valuable approach in the study of the structure and function of human skin. # 2002 Elsevier Science

B.V. All rights reserved.

Keywords: Aging; Photoaging; Collagen; Tryptophan

1. Introduction: background

The skin has been the subject of numerous studies

using optical spectroscopy beginning with the ele-

gant work of Edwards and Duntley [1]. Light in

skin can be either absorbed or scattered. Absorption

arises because photons interact with the various chro-

mophores found in skin, and scattering is due to

changes in index of refraction in the highly inhomo-

geneous nature of skin. The two most important skin

chromophores in the visible range of the spectrum

(400–700 nm) are hemoglobin and melanin. Certain

molecules (fluorophores), following absorption of light,

emit light at longer wavelengths as fluorescence. The

intensity of fluorescence emission is of the order of

1000 times smaller than the intensity of the incident

radiation.

Fluorophores are excited mainly in the ultraviolet

(UV) range of the spectrum and it is difficult, if not

impossible, to separate out the closely overlapping

absorption bands. In addition, numerous non-fluores-

cent chromophores absorb light in the UV range. The

principal UV absorbers in skin are melanin and hemo-

globin (330–400 nm), while at shorter wavelengths

(280–330 nm) the absorption of proteins becomes

dominant. Absorption also occurs due to a host of

other molecules such as nucleic acids (DNA and

RNA), NAD/NADH, urocanic acid, and others

[2,3]. It is interesting to point out that even though

melanin is strongly absorbing at wavelengths shorter

than 330 nm, its absorption in the skin is oversha-

dowed by the absorption of proteins. This has been

demonstrated by comparing the spectral absorption

of pigmented skin to the spectral absorption of non-

Vibrational Spectroscopy 28 (2002) 17–23

$ Presented in part at the conference, 25–30 June 2000,

Winnipeg, Canada.* Corresponding author. Tel.: þ1-908-874-1507;

fax: þ1-908-874-7205.

E-mail address: nkollia@cpcus.jnj.com (N. Kollias).

0924-2031/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved.

PII: S 0 9 2 4 - 2 0 3 1 ( 0 1 ) 0 0 1 4 2 - 4

pigmented skin [4]. The principal chromophores

found in skin are summarized in Table 1.

Another important issue to be considered in the

study of interaction of light with skin, is the penetra-

tion depth of the light. The depth to which radiation

penetrates the skin limits the depth from which infor-

mation can be obtained by employing an optical

technique in a non-invasive way. Penetration depth

is limited by the attenuation of light due to absorption

and scattering. There is a large body of in vitro and in

vivo data on the attenuation of UV–VIS radiation by

the components of the skin [2,5,6]. While the data are

reliable, the exact values of the attenuation coeffi-

cients as a function of wavelength depend to some

extent on the preparation of the tissue samples.

The penetration depth of light into the skin is about

1 mm at the red end of the visible spectrum (700 nm)

and it decreases by approximately an order of magni-

tude as we move towards the blue end (400 nm). It

then decreases even more by two orders of magnitude

between 400 and 300 nm. In this way, at 700 nm

information can be obtained about all skin layers,

but below 290 nm the effective penetration depth is

limited to the epidermis.

The amount of light arriving at any particular depth

in the skin may be estimated from the values of the

tissue’s optical properties (absorption, scattering). It

should be also kept in mind that the absorption of

radiation in skin can result in some specific biological

activity. In such a case it is possible to characterize the

effectiveness of each wavelength in terms of a specific

biological activity through an action spectrum. The

fluorescence intensity may also be strongly modulated

by absorbing chromophores, especially when there is a

significant overlap between the excitation and emis-

sion bands of a fluorophore with the absorption band

of a chromophore such as hemoglobin and melanin.

Changes in the fluorescence intensity may be

accounted for by changes in the quantum efficiency

(influenced by the environment) of the fluorescing

molecule, as well as by changes in its concentration.

It is convenient to study the excitation spectra, since

excitation bands tend to be narrower and correspond to

broad absorption bands.

1.1. Skin structure

The skin is a layered structure interspersed with

various appendages such as hair follicles, sweat glands,

and sebaceous glands, which we will ignore for the

purposes of this discussion. The outermost layer of the

skin is called the stratum corneum or horny layer and is

a part of the epidermis. This structure is made up of

about 10 cell layers with a total thickness of around

10 mm. The cells that make up the stratum corneum are

dead and flattened keratinocytes called corneocytes.

They have no nuclei and may be thought of as dried out

and flattened cell skeletons. Each one of these cells is

about 1 mm thick, is hexagonal in shape, and has a

planar diameter of 30–50 mm. The corneocytes are held

together by corneosomes, extracellular remnants of

desmosomes, and by lipids (waxes, free fatty acids,

Table 1

The principal chromophores of skina

Skin chromophore Spectral range of absorption Fluorescence Principal absorption maxima (nm)

Oxyhemoglobin UV–VIS NO 412, 542, 577

Deoxyhemoglobin UV–VIS NO 430, 555, 760

Melanin UV–VIS NO Monotonic increase to short wavelengths

Water IR-long VIS NO 760, 900, 1250, 1400, etc.

Porphyrins VIS Yes Ex: �405; Em: 600

Bilirubin VIS NO 460

NAD/NADH UV Yes Ex: �350; Em: 460

DNA/RNA UV NO 260

Tryptophan UV Yes Ex: 295; Em: 340–350

Urocanic acid UV NO 280

Collagen x-links UV Yes Ex: 335, 370; Em: 380, 460

Elastin x-links UV–VIS Yes Ex: 420, 460; Em: 500, 540

Keratin (dry), horn UV Yes Ex: 370; Em: 460

a NO: not observable; Ex: excitation wavelength; Em: emission wavelength.

18 N. Kollias et al. / Vibrational Spectroscopy 28 (2002) 17–23

triglycerides) and cholesterol. The stratum corneum is

like a sandwich wrap. It forms the barrier that keeps

water in and unwanted materials out of the body. Below

the stratum corneum lies the viable epidermis, which

consists of 10 cell layers. The epidermis is viable tissue

made up primarily of keratinocytes. In the epidermis

there is one melanocyte (a cell that makes melanin) and

one Langehans cell (antigen presenting, immune sys-

tem cell), for approximately every 45 keratinocytes.

T-cells may also be found in the epidermis following

an acute injury to the skin.

Melanin is made by the melanocytes in membrane

bound organelles called melanosomes. During a pro-

cess termed ‘‘melanization’’, melanosomes are trans-

ferred from the melanocytes to the keratinocytes. In

the epidermis there are nerve fibers but no blood

vessels. The epidermis is largely a cellular structure

and as such it does not have mechanical strength.

The strength of the skin comes from the dermis. The

epidermis is separated from the dermis by the base-

ment membrane. This membrane, when viewed in a

transverse section, undulates like a spring resulting in

areas where the epidermis is thinner and areas where

the epidermis is thicker. The capillaries come up into

the areas where the epidermis is the thinnest like

‘‘pegs’’ of dermis.

The dermis is made up primarily of collagen (about

70% of its dry weight). The collagen is organized into

microfibrils, the microfibrils into fibers, and the fibers

into bundles. It is the collagen bundles that can be

visualized in H&E stained histological sections of the

skin. The macroscopic structure of the collagen matrix

is the result of the cross-linking between the collagen

bundles, which form a ‘‘basket-weave’’-type (or chain-

link fence-type) structure that extends beyond the plane

into the third dimension. The collagen fibers and bun-

dles are surrounded by polysaccharides (glycosamino-

glycans), which form the water holding matrix in the

dermis. These are attached to the collagen bundles, thus

providing elasticity, and also act as a lubricant by

allowing relative motion of the bundles. The remaining

fibrous material in the dermis is elastin, which appears

in candelabra like structures (in young and not photo-

damaged skin) that extend up towards the epidermis.

The dermis also hosts a special group of cells, the

fibroblasts, which are responsible for the production

of collagen. Inflammatory cells may be found in the

dermis in the case of acute injury or disease.

2. Methods

The spectrofluorimeter used in the studies was

manufactured by SPEX Industries (Horiba Group,

Edison, NJ). It consisted of a double monochromator

based fluorescence spectrometer with a xenon lamp as

the excitation source. The radiation from the excita-

tion monochromator is focused into a quartz fiber

bundle with individual fiber diameters between 100

and 200 mm. The distal end of the fiber bundle is

Fig. 1. Schematic diagram of the experimental instrumentation. Radiation from the xenon lamp source is filtered by a double monochromator

at the excitation compartment (X) and is led through a fiber bundle to the skin site of interest. Emitted radiation is collected by optical fibers of

the bundle and is analyzed by a double monochromator in the emission compartment (M). The intensity of the emitted radiation is measured

by a photomultiplier (PMT). The output of the PMT is processed by a computer, which also controls the instrument.

N. Kollias et al. / Vibrational Spectroscopy 28 (2002) 17–23 19

brought into contact with the skin sample under study.

The delivery and collection fibers are packed together

in a random manner. An important feature of the

instrument is the double monochromator design,

which significantly improves the signal to noise ratio

at short wavelengths (l < 300 nm). A sketch of the

instrumental arrangement is shown in Fig. 1.

In general skin sites do not require any special

preparation for measurement. Occasionally, when

measurements were made on skin sites that were

particularly scaly, the site was pre-hydrated with a

piece of gauze for 10–20 min. Scale fluoresces

strongly when dry, but the fluorescence is quenched

when the stratum corneum is wet. The fiber bundle tip

was cleaned with an alcohol pad between measure-

ments. In some instances, a film of plastic was used

between the fiber bundle and the skin. The plastic film

was either made of TeflonTM or SaranWrapTM. The

latter attenuates the incident radiation by approxi-

mately a factor of two. When measurements were

made on hairless mice (SKH), the skin was gently

lifted from the animal’s back and was pulled across the

fiber bundle end. Slight pressure was exerted on the

skin to partially blanch it. The goal of this was to

minimize the effects of fluorescence signal attenuation

by endogenous hemoglobin.

3. Results and discussion

When measuring fluorescence, we have the choice

of acquiring either excitation spectra or emission

spectra. We chose to measure primarily excitation

spectra, because these also provide information on

the absorption properties of the chromophores. The

fluorescence excitation spectra from SKH hairless

mouse skin [7] indicate that there are three principal

excitation bands in the UV at 295, 335 and 370 nm.

The 295 nm band has been attributed to epidermal

tryptophan moieties and the other two bands have been

assigned to insoluble collagen cross-links. The

335 nm band is attributed to pepsin digestible collagen

cross-links (PDCCL) and the 370 nm band to collage-

nase digestible collagen cross-links (CDCCL).

In serial excitation scans where the emission wave-

length is incrementally increased by 20 nm, an iso-

emissive point appears in the spectra in the vicinity

of 315 nm (Fig. 2). On the short wavelength side of

315 nm, the fluorescence signals are of epidermal

origin and on the long wavelength side the signals

are of dermal origin. Tryptophan fluorescence has

been found in isolated or exposed dermis at inten-

sity levels that are similar to those of the epidermis.

We believe that the excitation radiation that induces

tryptophan fluorescence is attenuated by the epider-

mis before reaching the dermis. These conclusions

were drawn in experiments where skin samples were

taken from animals following sacrifice and the epi-

dermis was separated from the dermis. Fluorescence

spectra were then obtained from both epidermis and

dermis, while the tissues were kept moist wrapped in

paper towels wetted with physiological saline until the

measurements were carried out.

In human skin, we raised suction blisters by apply-

ing a negative pressure to a skin site of 8 mm in

diameter for approximately 30–40 min. This proce-

dure provided a gentle and constant force sufficient

to separate the epidermis from the dermis. The roof of

Fig. 2. Serial excitation spectra from the right cheek of a 27-year-

old volunteer. The excitation wavelengths range from 250 to

470 nm in increments of 10 nm. For each spectrum the excitation

monochromator was scanned from 250 to within 20 nm of the

emission monochromator setting. The emission wavelength of each

scan was incremented by 20 nm from the previous starting at

280 nm. The major fluorescence peaks have been labeled with the

corresponding excitation wavelength: (i) 295 nm excitation, due to

tryptophan fluorescence; (ii) 335 nm excitation, due to PDCCL;

(iii) 350 nm excitation, possibly due to NADH; and (iv) 370 nm

excitation, due to CDCCL.

20 N. Kollias et al. / Vibrational Spectroscopy 28 (2002) 17–23

the blister was cut and was folded over without

completely separating it from the skin. A piece of

aluminum foil was inserted under the blister top and

the blister top was replaced thus making sure that the

fluorescence measurements would be from the epi-

dermis alone. The blister roof was then folded over

and the dermis was exposed, the fluorescence of the

dermis was then measured. As in the case of the

animals, we found in humans that the 295 nm excita-

tion of tryptophan moieties fluorescence was observed

in the spectra from epidermis and from dermis with an

equally strong intensity. When the fiber probe was

spaced from the stratum corneum, we found a number

of weak fluorescence signals in the 340–400 nm range

that we believe originate from the epidermal keratins.

These signals are negligible when we measure with the

fiber bundle probe in contact with the skin. The

principal fluorophores in the UV are given in Table 2.

The NADH fluorescence appears always buried in

the collagen cross-link fluorescence. This is not sur-

prising, because the mitochondrial content of the

keratinocytes is relatively low as compared to other

tissue types where NADH fluorescence can be

observed (e.g. hepatocytes). We have also conducted

a number of experiments where the oxygen supply to

the skin was diminished by the application of a

pressure cuff, but we did not observe variations in

the NADH fluorescence.

There are numerous skin conditions that can be

examined and studied using fluorescence. We present

here some of those cases we have studied and also

some which seem to be of general importance for

future study.

3.1. Aging

In these series of experiments conducted on SKH

hairless mice we followed two groups of animals up to

the age of 18 months. The first group consisted of mice

that were 6-week-old and the second group consisted

of animals that were 6-month-old (breeders). The

experiment was repeated with additional groups of

animals. We found that the tryptophan fluorescence

(295 nm excitation) decreased with age, while the

PDCCL fluorescence (335 nm excitation) correspond-

ingly increased with age. The collagenase digestible

cross-link fluorescence (370 nm excitation) remained

essentially unchanged. For SKH hairless mice the

tryptophan fluorescence appears to reach a minimum

by 12 months of age and remains constant after that. In

measurements on humans we found that for persons

older than 60 years of age the intensity of tryptophan

fluorescence is reduced dramatically compared to

younger persons. In some cases it is altogether absent.

The elastin signal with excitation at 420 nm increases

with age (Fig. 3), which is in contrast to the observa-

tions on mouse skin, where this signal is very weak.

In human skin we also found that the collagenase

digestible cross-link fluorescence shifts to longer

wavelength with age (Fig. 3). It is interesting to note

that there are no inducible fluorophores with age,

i.e. there are no new fluorophores that are expressed

with age.

3.2. Proliferation

Tape stripping of the stratum corneum increases the

epithelial cell proliferation rate to compensate for the

lost corneocytes. We have shown [8] that tape strip-

ping increases the tryptophan fluorescence in a time

dependent manner. Therefore, the intensity of fluor-

escence at 295 nm excitation may be used as a marker

for cell proliferation. This is in agreement with similar

reports by other research groups using fluorescence as

a marker of proliferation [9]. In addition, we have

shown that tryptophan fluorescence is significantly

Table 2

The principal fluorophores of skin

Fluorophore Excitation wavelength (nm) Emission wavelength (nm) Intensity of signal

Tryptophan 295 340–350 Strong

Pepsin digestible collagen cross-links 335–340 380–390 Secondary

Collagenase digestible collagen cross-links 365–380 420–440 Strong

Elastin crosslinks 420 500 Weak

NADH 350 460 Weak

N. Kollias et al. / Vibrational Spectroscopy 28 (2002) 17–23 21

increased in psoriatic skin, which again is probably

due to proliferation [10]. Finally, it has been reported

[11] that inflammatory white blood cells exhibit char-

acteristic fluorescence patterns with excitation max-

ima in the 250–265 nm range. This fluorescence can

be exploited in the study of skin inflammation. Inflam-

mation is often connected with skin healing and

regeneration processes, which are in turn connected

to proliferation.

3.3. Photoaging

After prolonged exposure the UV part of sunlight

can cause significant damage on skin demonstrated as

acute sunburn. Chronic exposure produces elastosis or

even cancer. The effects of UV irradiation on skin can

be observed very early by the use of fluorescence

before the acute responses develop. The main obser-

vations are that tryptophan fluorescence increases

while PDCCL fluorescence decreases after exposure

[12,13]. The first phenomenon has been correlated to

the increase in epithelial cell proliferation induced by

UV irradiation, while the second has been attributed to

the UV-induced damage of collagen cross-links.

Therefore, skin fluorescence may be used as a marker

for photoaging.

3.4. Photobiological effects

The maxima of the excitation spectra indicate

wavelength specific absorption of the skin chromo-

phores and therefore irradiation at these wavelengths

may induce photobiological activity. In the UVB

region the spectral efficiency for producing an increase

in the fluorescence of skin resembles the fluorescence

excitation spectrum and has a maximum at the excita-

tion wavelength of tryptophan fluorescence (295 nm).

Irradiation at this wavelength increases skin fluores-

cence in both humans and mice [7,8]. On the contrary,

irradiation at the excitation wavelength of PDCCL

fluorescence (335 nm) decreases the intensity of this

maximum in mouse skin [12], but is not equally effec-

tive in human skin. It is interesting to note that the

295 nm maximum of absorption of tryptophan coin-

cides with the wavelength of maximal effectiveness of

UV radiation to produce erythema.

3.5. Fluorescence as a general monitoring tool

There are numerous other skin conditions, which

can be examined and characterized in a quantitative

way using fluorescence. We will briefly mention here a

few examples. (A) Fluorescence has been successfully

used to monitor the state of the utriculi in the rhino

mouse model (Fig. 4). The utriculi have been shown to

collapse with the application of retinoids and this

process can be monitored by fluorescence spectro-

scopy based on the fluorescence of the ‘‘horn’’ asso-

ciated with the comedones [14]. (B) Fluorescence

imaging and spectroscopy have been used in the

monitoring of acne condition in humans. In particular,

acne related bacteria have been observed to exhibit

characteristic coproporphyrin fluorescence [15,16].

(C) The same techniques can be used to study hyper-

pigmentation. UV fluorescence photography utilizes

Fig. 3. Serial excitation spectra from the right cheek of a 70-year-

old volunteer. The excitation wavelengths range from 250 to

470 nm in increments of 10 nm. For each spectrum the excitation

monochromator was scanned from 250 to within 20 nm of the

emission monochromator setting. The emission wavelength of each

scan was incremented by 20 nm from the previous starting at

280 nm. The major fluorescence peaks have been labeled with the

corresponding excitation wavelength. Compared to the same scans

from the younger individual of Fig. 2: (i) 295 nm excitation peak,

due to tryptophan fluorescence, is significantly reduced; (ii) 370 nm

excitation, due to CDCCL, appears to have shifted slightly to

longer wavelengths (380 nm excitation); and (iii) there appears

to be a shoulder at 420 nm excitation, possibly due to elastin.

22 N. Kollias et al. / Vibrational Spectroscopy 28 (2002) 17–23

collagen fluorescence and the increased melanin

absorption in the UV to enhance the contrast around

pigmented lesions [17].

3.6. Future

There are smaller fluorescence signals that have

not yet been characterized. Some are convoluted in

the spectra of stronger signals and require numeri-

cal deconvolution methods to be distinguished (e.g.

NADH mentioned above). A great challenge for fluor-

escence spectroscopy is to identify a characteristic

signal that can be used for accurate diagnosis of non-

melanoma skin cancer [18–20].

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Fig. 4. Excitation spectra from rhino mice treated for 2 weeks with

different concentrations of all-trans-retinoic acid (tRA): (a)

untreated; (b) 0.0001% tRA; (c) 0.001% tRA; (d) 0.01% tRA; (e)

0.1% tRA. The emission monochromator was set at 380 nm, while

the excitation monochromator was scanned from 260 to 360 nm in

increments of 2 nm. The spectra have been normalized at 260 nm

excitation. The induction of cell proliferation due to the application

of increasing concentrations of tRA is reflected by the correspond-

ing increase of the tryptophan fluorescence signal (295 nm

excitation), whereas the effectiveness of the treatment is demon-

strated by the decrease of the signal at 335 nm excitation.

N. Kollias et al. / Vibrational Spectroscopy 28 (2002) 17–23 23