In Vivo Fluorescence of Human Skin
A Potential Marker of Photoaging
David J. Leffell, MD; Mark L. Stetz;
Leonard M. Milstone, MD; Lawrence I. Deckelbaum, MD
\s=b\ Fluorescence is a feature of elastin and collagen,
both major compounds of human dermis that are altered
by age and photoexposure. We studied the intrinsic
fluorescence of skin in vivo in 28 human volunteers to
determine whether photoaging and chronologic aging of
the skin could be evaluated by this noninvasive tech-
nique. We demonstrate that the excitation of skin auto-
fluorescence by laser ultraviolet radiation yields charac-
teristic tissue fluorescence spectra that are unrelated to
age, pigmentation, or skin thickness. The differences in
skin autofluorescence appear to be related to photoex-
posure. Thus, laser-induced fluorimetry, a noninvasive
technique, may be adaptable as a marker of photoag-
ing.
(Arch Dermatol 1988;124:1514-1518)
Fluorescence is natural phenomenon of many
a
compounds13 in which light is absorbed at one
wavelength and emitted at a higher wavelength.
Individual chemical compounds have characteristic
absorption and emission spectra but to date in vivo
fluorimetry has had limited diagnostic use and has
not been applied to the skin. The diagnostic potential
of the fluorescence of malignant tissue after treat¬
ment with hematoporphyrin derivative has been
reported.4 There have also been reports of fluores¬
cence in dental caries5 and in atherosclerotic and
normal arteries.6-7 Fluorescent compounds in the skin
include elastin8 and collagen.9 In addition, it is known
that with age10 and exposure to ultraviolet radia¬
tion,11 these molecules may be altered. The mecha¬
nism by which ultraviolet radiation ages skin is
unknown, and, to a large extent, measurement of this
effect has been limited to in vitro qualitative micro¬
scopic analysis.12 This study defines the fluorescent
properties of skin in vivo and determines whether
age or sun exposure altered the spectral pattern of
fluorescence. By inducing fluorescence of skin and
evaluating the fluorescence spectra, we have been
Accepted for publication June 3, 1988.
From the Departments of Dermatology (Drs Leffell and Mil-
stone) and Medicine, Section of Cardiology (Dr Deckelbaum and
Mr Stetz), Yale University School of Medicine, New Haven, and
the Veterans Administration Medical Center, West Haven,
Conn.
Presented in part at the American Society for Dermatologic
Surgery Annual Meeting, Monterey, Calif, April 15, 1988.
Reprint requests to Department of Dermatology, Yale Universi-
ty School of Medicine, 333 Cedar St, New Haven, CT 06510 (Dr
Leffell).
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able to detect changes in the integument that result
from solar ultraviolet radiation exposure but do not
appear to relate to chronologic aging. Thus, skin
autofluorescence may serve as a marker for photoag¬
ing.
SUBJECTS AND METHODS
Twenty-eight volunteers consented to participate in a
study approved by the Human Studies Subcommittee of
the Veterans Administration Medical Center, West Haven,
Conn. The subjects were chosen on the basis of age (Table
1) to represent three stages in the aging process. Group 1
consisted of children with a mean age of 3 years without a
history of excessive sun exposure. Group 2, with a mean
age of 30 years, represented young adults who have had
routine exposure to sun in the northeastern United States
for most of their lives. Group 3, with a mean age of 63
years, included subjects with outdoor occupations who had
clinical evidence of solar damage.
Patient pigmentation was categorized according to the
criteria developed in the cooperative trials of psoralens
and ultraviolet A therapy.'3 The classification is summa¬
rized in Table 1. The study was conducted between the
months of November and March during which no subject
had significant tanning secondary to ultraviolet radiation
exposure.
All studies were performed in the Cardiac Laser
Research Laboratory at the Veterans Administration
Medical Center.
Skin fluorescence was induced by a helium-cadmium
laser (Omnichrome model 356-5MS) emitting radiation at
325 nm (ultraviolet light in the B range [UVB]) at 0.5 to 5
mW continuous-wave power (Fig 1). The laser was coupled
to the central fiber of a coaxial fiberoptic probe (EOTec
Corporation, West Haven, Conn) and placed 3 mm from the
skin surface over the buttocks, axillae, left temple, fore¬
head, and dorsa of the hands after wiping with alcohol.
Sun-protected sites included the buttocks and axillae.
Sun-exposed areas included the left temple, forehead, and
dorsa of the hands. Each of these sites represented region¬
al variation in dermal thickness.'4
Tissue fluorescence was transmitted by the coaxial fiber
bundle, focused into a spectrograph (Jarrell Ash Monospec
27), spectrally dispersed, and imaged onto an optical
multichannel analyzer (OMA, Princeton [NJ] Instruments
Inc). The OMA consisted of a linear diode array of 1024
discrete elements coupled to a microchannel plate intensi¬
fier. The detector simultaneously detected the fluorescence
intensity in the wavelength range 300 nm to 700 nm. Ten
33-ms scans per reading were averaged. The OMA system
was spectrally calibrated using a mercury vapor lamp and
radiometrically calibrated using a National Bureau of
Standards traceable calibrated white light source (Uptron-
ics Model 245A). The latter calibration corrects for any
nonuniformity in the spectral sensitivity of the system.
The fluorescence spectrum was digitized using the diode
 array signals and the data were stored and analyzed using
a microcomputer (IBM PC AT). Table 1.—Groups of Subjects*
To further evaluate skin autofluorescence the fluores¬
Group N Age, y/Sex Skin Type Mean Age
cence of dermal compounds was studied in vitro. Using the
1 1.3/F I
system described above, elastin from bovine nuchal liga¬
ment and type I collagen from bovine tendon (Sigma 0.5/M
Chemical Co, St Louis) were studied. Desmosine and 4/M
isodesmosine (Elastin Products, St Louis), cross-linking 4/M 2.96 ± 0.06
amino acids found exclusively in elastin were examined as 3.3/F
well. All compounds were studied in the solid, nonsolubil- 3/F
ized form. 3/F
To study the fluorescence of human dermis in vitro, and 13 28/M
correlate these findings with the in vivo observations, an 24/M
elastin extraction technique was used. Solar-exposed (fa¬ 27/M IV
cial) and solar-protected (buttock) skin was obtained from 26/M
two patients. The skin was cut into 1-mm sections, placed
in saline, and autoclaved at 121 °C and 1 atm of pressure for 26/M
four hours.'5 The fluorescence of the residual tissue was 31/M
then studied using the system described above. In addition, 31/M 29.6 ± 0.83
the specimens were stained for elastin using the metachro- 35/M
matic Richardson stain and studied by light microscopy. 31/M
Statistics 32/M
31/M
The spectral values for solar-exposed and solar-pro¬ 31/F
tected skin were compared using the Wilcoxon signed-rank
31/M
test. The Kruskal-Wallis test was used to determine
whether the ratios for solar-exposed skin in all three 59/M
56/M
groups were the same and whether the ratios for solar-
protected skin were the same in groups 1, 2, and 3. All 56/M
values are expressed as mean ± SEM. 65/M 63 ± 2.0
67/M
RESULTS
65/M
Twenty-eight subjects were evaluated. The fluo¬ 63/M
rescence pattern of sun-protected skin all three age 74/M II
groups was similar suggesting that age was not an 'Group indicates the experimental group to which subjects were
interesting variable. In group 1, the fluorescence assigned based on age; N, number of subjects in each group; skin type:
pattern of sun-exposed skin did not vary significant¬ I, never tan, always burn; II, always burn but sometimes tan; III,
ly from that of sun-protected skin. Figure 2 (top) sometimes burn, but always tan; IV, never burn, always tan; V,
demonstrates a typical spectrum that was seen in moderately pigmented; VI, blacks; mean age, years ± SEM.
this group of subjects who have had minimal cumu¬
lative environmental ultraviolet radiation exposure.
The shoulder or second peak at 429 nm was charac¬ The results of the in vitro analysis of dermal
teristic of solar-protected skin in all groups but was compounds are represented in Fig 4. Both desmosine
present in solar-exposed skin in group 1 only. In and elastin are intensely fluorescent with a peak of
contrast, subjects in groups 2 and 3 showed a distinct intensity at 390 nm. Collagen type I demonstrated
difference in the fluorescence spectra of sun- peak fluorescence intensity similar to that of desmo¬
protected and sun-exposed skin. (Fig 2, bottom, and sine but it occurred at 378 nm. The similarity of the
Fig 3). elastin and desmosine curves is evident as is their
The presence of the two peaks of fluorescence variation from that of collagen type I.
intensity noted at 390 nm and 429 nm was used to In vitro analysis of human-derived solar-exposed
quantitatively compare spectral patterns. A ratio of and sun-protected dermal tissue demonstrated spec¬
fluorescence intensity at 390 nm to 429 nm (R390/ tra similar to the corresponding in vivo pattern. The
429) was calculated for each skin spectrum. The SE R390/429 for solar-exposed skin was 1.00 and 1.03
of the ratio reflects individual variation in the and that for solar-protected skin was 1.25 and 1.21,
spectra. respectively. Histologie evaluation of the specimens
In group 1, there was no difference between sun- revealed amorphous tissue devoid of epidermis that
protected and sun-exposed skin in R(390/429) stained primarily for elastin.
(1.19 ± 0.06 and 1.15 ± 0.06, respectively) (Table 2). COMMENT
In group 2 R(390/429) of sun-protected skin was
1.14 ± 0.04 and that of sun-exposed skin was 0.88 ± Interest in the biology of aging continues to
0.02 (P < .001). A similar difference between solar- expand. One physical agent that appears to play a
protected and solar-exposed skin was found in group role in the aging process of skin is ultraviolet radia¬
3(1.11 ± 0.04 vs 0.91 ± 0.04; P < .008). Solar-exposed tion. To date, however, it has been difficult to
skin of groups 2 and 3 differed significantly from all evaluate photoaging in a noninvasive fashion.
other groups (P < .002). We describe here the observation that solar-

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Helium Cadmium Laser
D 0
UV
Transmitting
CRT Display
/\
Controller
Computer
Fig 1.—Laser apparatus for induction of skin fluorescence (see "Subjects and Methods" section for detailed
explanation).
UV indicates ultraviolet light and CRT, cathode ray tube.
exposed skin has a different inducible fluorescence
pattern than that of solar-protected skin. The simi¬
larity of solar-exposed and solar-protected skin spec¬
tra in the youngest age group is consistent with the
fact that these children have not yet had much sun
exposure. Conversely, subjects in groups 2 and 3
showed significant differences in the fluorescence of
buttocks and axillae when compared with the temple
or other solar-exposed areas. That the differences
observed in groups 2 and 3 were not simply a
function of regional variation in skin thickness is
confirmed by the fact that spectra of the buttocks
were similar to those of the axillae—another sun-
protected region of the body with different morphol¬
ogy.14 In addition, the fluorescence spectra of skin
from the shoulders were similar to those of the
forehead despite the consistently thicker dermis of
the former sites.16
The fact that the greatest difference in fluores¬
cence exists between groups 1 and 2, when clinically
one might have expected it to occur between groups 2
and 3 suggests that the changes in dermal composi¬
tion reflected by fluorescence activity may be sub-
clinical, occurring early in the evolution of photoag¬
ing.
The majority of our subjects were of skin types I
and II and tended not to tan. They therefore may
have had more severe photoaging compared with
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Lens
Single Fiber
Filter
Coaxial Fiberoptic
Catheter
Fiber Bundle
Achromatic Lens
Spectrograph
Diode Array Detector
those patients with darker pigmentation. The pres¬
ence of melanin in the epidermis may attenuate the
fluorescence intensity at 390 nm thus altering the
R390/429. Experimentally, the pattern of the fluo¬
rescence spectra of those patients with skin types III
and IV were similar to those of patients with less
melanin although, as expected, the overall intensity
of the curves was diminished. The observation of
R390/429 typical for solar-exposed and solar-pro¬
tected in in vitro skin lacking melanin suggests that
this system may be sufficiently sensitive to detect
solar-induced changes despite the potential reab-
sorption artifact related to this pigment.
The anatomic source of the observed in vivo fluo¬
rescence remains to be determined. Studies1' with
human cadaveric aorta suggest that fluorescence,
when induced by ultraviolet radiation, appears to
derive from tissue at a depth of 150 nm to 200 nm.
While aorta has a different composition than human
skin and the fluorescence spectrum is different, this
observed depth of fluorescence appears to correspond
with preliminary studies performed by us in human
mastectomy skin in vitro. Since human epidermis is
75 to 100 nm thick in all areas except the palms and
soles,18 and the dermis varies in thickness from 700 to
1500 nm,"' it is possible that the in vivo induced
fluorescence in human skin derives from excited
compounds in the papillary dermis. Interestingly, it
 2600
Nonsolar Skin --Nonsolar Skin
—Solar Skin
—
Solar Skin

2000-
3
sí

300 400 500 600 700

Wavelength, nm
800
-Nonsolar Skin
700 Solar Skin i i i i i i i i i i
300 350 400 450 500 550 600 650 700
600 Wavelength, nm

500 Fig 3.—In vivo laser-induced fluorescence spectra of skin.

400 Subject from group 3. Nonsolar skin refers to fluorescence
spectrum of buttocks. Solar skin refers to fluorescence spec¬
300 trum of left temple and forehead.
200
100
Table 2.—Ratios of Fluorescence Intensity
i i i i i i i i
' '
Solar-Protected vs Solar-Exposed Skin*
300 400 500 600 700
Wavelength, nm Group Nonsolar Solar
Fig 2.—In vivo laser-induced fluorescence spectra of skin. 1 1.19 ± 0.06 1.15 ± 0.06 NS
Nonsolar skin refers to fluorescence spectrum of buttocks and 1.14 ± 0.04 .88 ± 0.02 .001
solar skin to fluorescence spectrum of left temple and fore¬ 1.11 ± 0.04 .91 ± 0.04 .001
head. Top, Subject from group 1. Both spectra have a peak at *
Solar refers to sun-exposed skin and nonsolar refers to sun-
390 nm and additional shoulder at 429 nm. Spectra are typical protected skin; ratios represent fluorescence intensity of subject's skin
of other subjects in this group. Bottom, Subject from group 2. at wavelength 390 nm divided by the fluorescence intensity at
Spectrum of sun-protected skin is similar to sun-exposed and wavelength 429 nm; values, mean ± SEM; and P, Wilcoxon signed-
sun-protected spectra of 4-year-old subject above. Fluores¬ rank test comparing solar and nonsolar ratios in each group.
cence spectra of sun-protected and sun-exposed skin differ.

6000 •Desmosine
---Collagen
is in the papillary dermis that important photo- 5000 -Elastin
induced changes in elastin and collagen are noted.19
The quantitative fluorescence data reported here 4000
appear to relate more to photoinduced changes than
3
>í
to chronologic aging but the precise source of the « 3000-
fluorescence that we have observed remains to be c
Sic
elucidated. In light microscopy, it is often the colla¬
2000
gen that is thought to autofluoresce. Importantly,
collagen, despite its slow turnover rate in adults does
change with age.2021 With photoexposure, the amount 1000
of mature collagen decreases.22 Elastic tissue in skin12
and aorta23 changes with age and in the skin with 0
300 350 400 450 500 550 600 650
exposure to ultraviolet radiation.24 The changes in 700
elastic tissue in skin due to ultraviolet radiation are Wavelength, nm
different from those due to chronologic age alone.12 Fig 4.—In vitro fluorescence spectra of elastin, desmosine, and
Since it is known that ultraviolet-induced changes collagen. All compounds were studied in solid, nonsolubilized
in elastin are different from those due to age alone, it state.
is interesting to consider that components of elastic
tissue may be responsible for the observed changes
in the fluorescence pattern. While photodamaged therefore deserve further study. LaBella and Lind¬
skin may contain more elastin than sun-protected say23 suggested that the major fluorophore in elastin
skin, the biochemical nature of this elastotic materi¬ was probably a cross-linking agent because fluores¬
al is different from elastin itself.24 The possibility cence was greatest in those peptides resistant to
that changes in fluorescence pattern due to sun degradation by elastase. One of the major cross-
exposure relate to elastin in one form or another may linking amino acids of elastic tissue, desmosine, has

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 been demonstrated to increase fourfold in sun-
exposed skin compared with sun-protected skin.25 In
addition, Lamy et al26 have demonstrated that des¬
mosine is a photoreactive amino acid. Elastin and
desmosine have a similar fluorescence spectral pat¬
tern to that of human skin in vivo, and share a peak
of intensity at 390 nm while the spectrum of collagen
has a peak of intensity at 378 nm and has a grossly
different shape compared to skin, elastin, and des¬
mosine. Further studies into the role of these com¬
pounds in the in vivo phenomena described here may
be warranted.
It should be noted that fluorescence spectra for
compounds in vivo may be altered compared with the
spectra of those same compounds in the crystalline
state or in solution. The fluorescence patterns of the
dermal compounds studied may be altered by the pH,
water content, and tertiary structural arrange¬
ment.8
The preliminary study with human-derived der¬
mal tissue suggests that the differential fluorescence
pattern of solar-exposed and solar-protected skin is
preserved in vitro. Moreover, the absence of epider¬
mis in these specimens suggests that epithelium or
its constituents do not contribute significantly to the
observed fluorescence spectrum. Although com¬
pounds present in the epidermis such as melanin,
nucleic acids, or small aromatic compounds could
absorb dermal fluorescence in a differential fashion
and alter the recorded spectrum of our studies of
human skin, in vitro studies suggest that this may
not be an important factor.
The use of R390/429 to compare spectra between
groups was determined by the observation of natu¬
rally occurring peaks of fluorescence intensity. Other
methods of pattern analysis may permit more specif¬
ic quantitative analysis of cutaneous fluorescence.
The use of skin fluorescence to study changes in
the skin as they relate to effects of ultraviolet
radiation has important implications for under¬
standing the pathogenesis of photoaging. The rapid
and versatile noninvasive fluorimetric studies of
human skin using the system described here may
permit prospective in vivo evaluation of solar-
induced aging in this most accessible organ. The
sensitivity of the tissue fluorescence technique may
aid in the identification of molecular changes associ¬
ated with photoaging. While determination of the
precise usefulness of this technique to quantitate
this process and its biologic correlates awaits further
study and refinement, the observation of in vivo
tissue fluorescence may be utilized to develop a
marker of photoaging.
This study was supported by grants from the American Society
for Dermatologie Surgery, Evanston, 111; the Dermatology Foun¬
dation, Evanston, 111; the American Heart Association, Connecti¬
cut Affiliate Inc; and US Public Health Service grant No. 5-
T32-AR07016.
We thank Aaron B. Lerner, MD, and Irwin M. Braverman, MD,
for their critique of this manuscript; Gary L. Rosner, ScD, for his
statistical evaluation of the data, and our colleagues and their
children who volunteered for this study.
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