Journal of Biomedical Optics 13共4兲, 041320 共July/August 2008兲

Risk estimation of skin damage due to ultrashort pulsed,

focused near-infrared laser irradiation at 800 nm
Frank Fischer Abstract. New imaging techniques using near-infrared 共NIR兲 femto-
Research Microscopy second lasers 共fs-lasers兲 in multiphoton laser scanning microscopy
Beiersdorf AG 共MPLSM兲 have great potential for in vivo applications, particularly in
Unnastrasse 48
D-20245 Hamburg, Germany human skin. However, little is known about possible risks. In order to
evaluate the risk, a “biological dosimeter” was used. We irradiated
fresh human skin samples with both an fs-laser and a solar simulator
Beate Volkmer UV source 共SSU兲. DNA damage introduced in the epidermis was
Dermatologisches Zentrum Buxtehude evaluated using fluorescent antibodies against cyclobutane-pyrimidin-
Am Krankenhaus 1 dimers 共CPDs兲 in combination with immunofluorescence image
D-21614 Buxtehude, Germany
analysis. Four fs-irradiation regimes 共at 800-nm wavelength兲 were
evaluated differing in laser power and step width of horizontal scans.
Stefan Puschmann Fs-irradiation did not give CPDs at 15-mW or 30-mW irradiation
Research Microscopy power using 10 horizontal scans every 5 microns. CPDs could be
Beiersdorf AG seen at 60-mW laser power and 5-␮m step size and at 35-mW using
Unnastrasse 48 1-micron step width. Quantitative comparison of SSU-induced CPDs
D-20245 Hamburg, Germany showed that the 60-mW laser irradiation regime is comparable to
UV-irradiation, giving 0.6 minimal erythemal dose 共MED兲. The
Ruediger Greinert 1-micron irradiation regime was comparable to 0.45 MED. Under
Wolfgang Breitbart these experimental conditions, the risk of DNA damage due to fs-laser
Dermatologisches Zentrum Buxtehude irradiation on skin is in the range of natural UV-exposure. © 2008 Society
of Photo-Optical Instrumentation Engineers. 关DOI: 10.1117/1.2960016兴
Am Krankenhaus 1
D-21614 Buxtehude, Germany
Keywords: multiphoton processes; microscopes; laser applications; imaging;
llumination; high-power lasers.
Juergen Kiefer Paper 07264SSRR received Jul. 18, 2007; revised manuscript received Apr. 18,
University Giessen 2008; accepted for publication May 13, 2008; published online Aug. 27, 2008.
Strahlenzentrum
Leihgesterner Weg 217
D-35392 Giessen, German

Roger Wepf
Research Microscopy
Beiersdorf AG
Unnastrasse 48
D-20245 Hamburg, Germany

1 Introduction ment and studies could be related to the fact that little is
known about the risk of this type of laser application in vivo,
A few decades ago, ultrashort pulsed laser sources, e.g., although cell damage has been shown.10 Damage thresholds
femtosecond–laser 共fs-laser兲 systems, became available.1,2 But for in vitro applications can be found.11,12 For example, the
it was only recently that they found interest in biomedical laser power leading to cell damage in microscopy applications
research.3 They possess potentials for a multitude of new was found12 to start with irradiances as low as 2 mW. The
methods and techniques in different fields of biomedical re-
threshold power depends on pulse duration, mean power, and
search, like controlled cell damage in the micrometer range,4
peak power.11
nanosurgery,5,6 multiphoton laser scanning microscopy
Using ultrashort pulsed, focused laser irradiation in the
共MPLSM兲,2,4 DNA-protein cross-linking,6,7 and optical coher-
near-infrared 共NIR兲 wavelength range, it is possible to reach
ence tomography 共OCT兲.8 So far, these techniques were ap-
plied in routine use in vivo only for laser surgery of the cor- deeper skin layers 共e.g., the stratum germinativum兲 compared
nea, although some other in vivo applications seem to be to UV.13 Due to the nonlinear effects of ultrashort focused
possible and desirable.9 laser irradiation, biologically relevant skin chromophores are
One reason for the delayed development of in vivo equip- excited comparable to one-photon excitations in the UV
range. If the irradiance is high enough, two or more photons
can be absorbed simultaneously. Two-photon absorption
Addresss all correspondence to: Frank Fischer, PhD, PO Box 518, Beiersdorf
AG, Unnastrasse 48, D-20245 Hamburg, Germany. Email:
[email protected]. 1083-3668/2008/13共4兲/041320/8/$25.00 © 2008 SPIE

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 Fischer et al.: Risk estimation of skin damage due to ultra-short pulsed, focused near-infrared irradiation…

Fig. 1 Sample preparation and irradiation procedure. 共a兲 Biopsy is divided and irradiated using fs-irradiation 共left兲 or UV-irradiation 共right兲.
共b兲 Fs-irradiation regime 1to 3 = 10 horizontal scans around the basal membrane; fs-irradiation regime 4 = 150 horizontal scans from the skin surface
to the dermis.

共2PA兲 and three-photon absorption 共3PA兲 allow excitation of fluorescent-labeled CPD antibodies can be used as a measure
higher energy levels in tissue chromophores. For example, for laser-induced CPD formation. We compared CPDs
800 nm reaches energy states that are usually accessible only induced by 1.5 minimal erythema dose 共MED兲 of solar UV.
by 400-nm 共2PA兲 or 267-nm 共3PA兲 irradiation. Tissue chro-
mophores like flavins, porphyrines, and/or lipoproteins can be 2 Materials and Methods
excited by two photons. Simultaneous absorption of three
2.1 Skin Samples
800-nm photons can excite DNA, NADH, collagen, proteins,
and other tissue molecules. Skin samples were obtained from healthy volunteers 共photo-
Because of the low pulse energy in the nanojoule range types II to III兲 after written consent. Each of eight volunteers
and the low average power 共milliwatt兲 of fs-laser irradiation gave two skin biopsies, 6 mm in diameter, one from the neck
used in biomedical research, especially in MPLSM, all effects and one from the buttock. Samples from the neck were in-
on tissue are connected with nonlinear effects, which occur cluded in this study in order to be able to distinguish effects of
due to the high peak irradiance, which reaches several hun- extended UV irradiation due to sun exposure from fs-NIR-
dred GW/ cm2. Photochemical damaging induced by linear irradiated skin. After excision, the skin samples were cut in
absorption, which can also occur at low irradiances, is not half.
expected to play a role in the wavelength range between
700 nm and 1300 nm, which is usually used with fs-lasers. 2.2 Irradiation
With the exception only of melanin, the endogenous chro- Before skin sample excision, the sensitivity of the volunteers
mophores do not absorb in this wavelength range.14–17 There- to UV-irradiation was quantified. One minimal erythema dose
fore, in tissue with low melanin content, the risk of damage 共MED兲 was evaluated by visual assessment of six spots irra-
due to one-photon absorption 共1PA兲 is expected to be neglect- diated using increasing radiation power of the UV source. The
ably low. Only biostimulation effects of low-irradiance NIR erythema was defined according to COLIPA 共The European
radiation 共below 300 mW/ cm2兲 were described.18–22 Cosmetic Toiletry and Perfumery Association兲 as the first per-
Therefore, only two mechanisms are expected to be rel- ceptible, clearly defined, unambiguous reddening of the skin
evant for tissue damage, photochemical effects caused by 24 h after irradiation. UV- and fs-irradiations were performed
multiphoton absorption and optical breakdown. It is well on freshly excised samples 共maximum 1 h after excision; see
known that blue and UV light irradiation is able to damage Fig. 1兲.
DNA and/or other biomolecules, which absorb in this wave- For the UV-irradiation, the whole surface of one half of the
length range. In skin, photochemical damage results in imme- sample was irradiated using a solar simulator 共SU 5000, mut
diate tissue response 共e.g., sunburn兲 and long-term effects like GmbH, Hamburg, Germany兲 at a UV dose according to 1.5
photoaging or even skin cancer.3,14 MED. The UV-irradiation took about 15 min. After the UV-
Little is known about the action of fs-lasers in skin. Meth- irradiation, the samples were frozen in liquid nitrogen for fur-
ods based on “biological dosimetry” are the only option to ther use. A spectrum of the solar simulator emission is shown
observe biological effects. Some dosimetric methods use the in Fig. 2.
sensitive and quantitative assay of cyclobutane-pyrimidine- The other half of the skin sample was irradiated using an
dimers 共CPDs兲 in DNA visualized by immuno- ultrashort pulsed, focused laser beam of a Mai-Tai laser
fluorescence detection.23 Cell aberrations may result if in- 共Spectra Physics, Darmstadt, Germany兲 at a wavelength of
duced CPDs are not repaired or are misrepaired by the poly- 800 nm, using a repetition rate of 80 MHz and a pulse width
merase repair system.24,25 The fluorescence intensity of the at sample layer of about 150 fs. For scanning and focusing

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 Fischer et al.: Risk estimation of skin damage due to ultra-short pulsed, focused near-infrared irradiation…

images can be taken with good contrast. At 60 mW, first dam-

aging occurs during scanning 共fluorescent scars, as described
elsewhere4兲. The fourth regime covered the whole depth from
the surface to the papillary dermis using linearly increasing
laser power 共Fig. 1兲. The most suitable laser power to achieve
best imaging results was used in order to simulate tissue im-
aging as one possible in vivo application.
The four irradiation regimes were applied side by side on
the second half of the original skin sample, producing irradi-
ated areas of about 200⫻ 200 ␮m. Between irradiated areas,
a space of 400-␮m unirradiated tissue was left in order to
avoid cross talk between irradiated areas due to light scatter-
ing in the skin. At each side of the row of the four irradiated
Fig. 2 Emission spectrum of the solar simulator SU 5000.
areas, the skin was marked with a black spot in order to re-
trieve the tiny irradiated areas for the subsequent CPD analy-
sis. The whole fs-irradiation took about 15 to 20 min;
samples were frozen in liquid nitrogen at −196 ° C after fs-
purposes, a MPLSM 共DermaInspect, Jenlab, Jena, Germany兲 irradiation for further analysis.
equipped with a Zeiss Plan/Neofluar 40⫻ NA 1.3 objective
lens 共oil immersion兲 was used. The DermaInspect device con-
tains a laser emitting NIR in the range between 750 and
2.3 Cyclobutane-Pyrimidin-Dimer Antibodies and
850 nm according to the specification by the manufacturer.
The focus of the laser beam was scanned at various depths
Staining Protocol
within the epidermis and papillary dermis of the excised half Sections 共7 ␮m thick兲 were cut from frozen tissue using a
of the skin sample. Each horizontal scan was 200 cryostat 共Microm, Walldorf, Germany兲 and were sequentially
⫻ 200 microns wide and took 32 s. Automatic horizontal fixed after slide preparation for 5 min in methanol and ac-
scanning 共parallel to the skin surface兲 using a fixed step width etone at −20 ° C. After fixation, the slides were stored at
in the depth of the microscope focus was performed. Four −20 ° C. For CPD staining, the samples were rehydrated in
laser irradiation regimes were chosen 共Fig. 1兲: TKT 100 共50 mM Tris pH 7.2; 1 M KCl, 0.3% Triton X-100兲
• 10 horizontal scans at 15 mW laser power 共96 J / cm2 for 60 min at room temperature, washed once in phosphate
peak fluence, 0.64 TW/ cm2 peak irradiance兲, 5 microns in buffered saline 共PBS兲 for 5 min and two times for 5 min in
depth apart from each other; 2⫻ SSC 共300 mM NaCl, 30 mM Na citrate兲. The sections
• 10 horizontal scans at 30 mW laser power 共191 J / cm2 were denatured in 0.1 N NaOH/70% EtOH for 3 min; dehy-
peak fluence, 1.28 TW/ cm2 peak irradiance兲, 5 microns in drated for 1 min each in 70%, 90%, and 100% EtOH; air
depth apart from each other; dried; and incubated with Proteinase K 共10 ␮g per ml; Sigma,
• 10 horizontal scans at 60 mW laser power 共383 J / cm2 Taufkirchen, Germany兲 at 37 ° C for 10 min. After three
peak fluence, 2.55 TW/ cm2 peak irradiance兲, 5 microns in washes for 5 min each in PBS, they were incubated with 5%
depth apart from each other; goat serum 共Dako, Hamburg, Germany兲 for 30 min at room
• 150 horizontal scans at linear increasing laser power temperature, rewashed three times for 5 min in PBS, and in-
with depth 共2 mW to ca. 35 mW, 13 to 223 J / cm2 peak flu- cubated overnight at 4 ° C with monoclonal antibody specific
ence, 0.085 to 1.5 TW/ cm2 peak irradiance兲, 1 micron in for CPD 共Kamiya Biochemical, Seattle, Washington兲 diluted
depth apart from each other. Power was increased linearly, 1:1000 in PBS. Sections were then washed three times for
since image quality was found to be best when linearly in- 5 min each in PBS and incubated with goat anti-mouse IgG
creasing the power with depth. conjugated with fluorescein isothiocyanate 共FITC; Dianova,
Peak fluence 共or peak radiant fluence兲 is the time integral Hamburg, Germany兲 diluted 1:100 in PBS. After three addi-
of the spherical peak irradiance; it combines the two main tional 5-min washes in PBS, slides were mounted with “an-
paramters influencing dosimetry and hence photochemical ef- tifade” 共2.3% DABCO; Sigma, Taufkirchen, Germany兲 in
fects, in our case, the irradiance and the time. The wavelength 90% glycerol in 20 mM Tris, pH 8.0 and covered. Sections
and the irradiation time were kept constant during experi- were visualized using a CCD camera 共Kappa, Gleichen, Ger-
ments in order to investigate the effects of one parameter, the many兲 coupled to a fluorescence microscope 共Leica DM,
peak irradiance. Leica, Wetzlar, Germany兲. Fluorescence intensity was quanti-
The first three regimes were applied around the basal fied using a digital imaging system 共OPTIMAS, Bothell,
membrane in order to make sure that the most sensitive cells Washington兲. Under the premise that the images of the differ-
for risk evaluation, the basal layer cells, will receive the laser ent samples were taken under the same camera conditions and
light. Three different laser powers 共15, 30, and 60 mW兲 were a careful handling of the probes to minimize the influence of
applied, because with tissue imaging, these laser powers had photobleaching, the fluorescence intensity is a reliable mea-
been shown to cover the possible working range when imag- sure for the amount of induced CPDs 共e.g., Refs. 26 and 27兲.
ing horizontal scans at basal membrane depth. Laser power Comparing the data of laser-induced CPDs with those induced
was measured using a power meter 共Ophir Laserstar兲 with a by 1.5 MED solar UV radiation 共UVR兲, the method can be
coverglass and immersion oil in front of the detector. In this used to establish the possibility for estimation of skin cancer
depth, at 15 mW, imaging starts to be possible. At 30 mW, risks according to current models.

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 Fischer et al.: Risk estimation of skin damage due to ultra-short pulsed, focused near-infrared irradiation…

3 Results Table 1 Average and standard deviation of the relative amount of

CPDs per nucleus for UV irradiation 共1.5 MED兲 on buttock skin and
First, an astonishing by high amount of CPD-related fluores- fs-irradiation on buttock and neck skin.
cence could be found in unirradiated neck tissue, indicating a
recent UV exposure of volunteers. An example using green Average SD
fluorescent CPD antibodies is shown in Fig. 3共a兲. For com- fluorescence fluorescence
parison, a CPD-stained buttock tissue slice is shown in Fig. Treatment intensity/nucleus 关a.u.兴 intensity/nucleus 关a.u.兴
3共b兲. Apart from the nuclei, the images exhibit a low overall 1.5 MED-UV 58.4 19.1
fluorescence in the tissue due to a slight unspecific binding of
the antibody. While in the neck skin 关Fig. 3共a兲兴 almost all cell Fs 共neck兲 27.9 7.3
nuclei show green fluorescence, the nuclei of buttock tissue
Fs 共buttock兲 24.8 8.0
are black 关Fig. 3共b兲兴. Hence, UV-induced CPDs could already
be detected in unirradiated neck tissue 共due to recent solar UV Fs 共buttock 2兲 18.1 2.1
exposure兲, while the buttock skin cells show no damage.
Due to the high amount of CPDs found in native neck
tissue 共not irradiated during the experiment protocol兲, a com-
parison between unirradiated and irradiated neck tissue in or- found in unirradiated areas of the neck tissue as well, prob-
der to quantify changes due to fs-laser irradiation was not ably due to recent solar exposure of the volunteer 关see Fig.
possible 共data not shown兲. In order to compare the effect of 4共a兲兴. The areas where fs-irradiation was applied could not be
the different irradiation regimes 共unirradiated, UV-irradiated, distinguished from the surrounding unirradiated tissue. Hence,
and fs-laser irradiated兲 only buttock tissue was evaluated. the numbers given in Fig. 4 and Table 1 for fs-irradiated neck
Examples are shown in Fig. 4. tissue may be mainly ascribed to CPDs found in native tissue.
In the unirradiated buttock samples of all participating vol- The fs-irradiation has little or no additional effects in this
unteers, no CPD fluorescence could be detected 关Fig. 4共a兲兴. In case. Neck tissue values are given here only for comparison in
contrast, many nuclei of the solar simulator irradiated buttock order to be able to classify sun-exposed tissue in terms of
tissues 共UV dose equivalent to 1.5 MED兲 showed CPD fluo- CPD induction.
rescence with high fluorescence intensity in all volunteers Buttock skin irradiated at 15-mW and 30-mW fs-laser
关Fig. 4共b兲兴. In the fs-irradiated buttock tissue of all volunteers, light using 10 horizontal scans around the basal membrane
spatially confined areas of CPD fluorescence could be found did not show any CPDs. At 60-mW fs-irradiation, 42% of the
关Figs. 4共c兲 and 4共d兲兴. Horizontal dimensions of these areas CPDs observed in 1.5 MED UV-irradiated buttock skin were
were about 150 ␮m, which is about the field of view of the in found 共Table 1兲. Thus, the amount of CPDs induced by
vitro multiphoton microscope setup used. With the known lo- 60-mW fs-irradiation is equivalent to a dose of about 0.6
cation of the irradiation regimes due to the marking used on MED for that small irradiated area.
the skin, it seems safe to assume that this fluorescence origi- Irradiation regime four 共2 to 35 mW兲 resulted in 30%
nates from the CPDs produced using 60-mW fs-irradiation. CPDs compared to the 1.5 MED UV irradiation. Therefore,
Additionally, in three buttock samples, a second area with this irradiation regime gave 0.45 MED. The fs-irradiated area
CPD fluorescence could be found 关Fig. 4共d兲兴. CPD fluores- was much smaller than the UV-irradiated area, resulting in
cence in this area was lower and restricted in the horizontal much smaller amounts of fs-irradiated cells.
dimensions to a small area. 共Only four nuclei show fluores-
cence signal.兲 This fluorescence is assumed to be due to the 4 Discussion
femtosecond irradiation regime applied at varying power from
2 to 35 mW every 1 ␮m in depth. 4.1 UV- and Fs-NIR-Irradiation Effects in Human Skin
A quantitative analysis of the CPD fluorescence found in In the unirradiated areas of fs-exposed samples and untreated
UV-irradiated and fs-irradiated buttock skin for all eight skin buttock samples, no CPD-related fluorescence could be ob-
samples is given in Fig. 5. In order to quantify the induced served 关Figs. 4共a兲 and 4共c兲兴. In contrast, unirradiated areas of
DNA damage, the fluorescence intensity of up to 40 nuclei per fs-exposed neck samples showed already a high amount of
slice was measured 关arbitrary units兴 and average values for CPDs, corresponding to 48% of CPDs induced by a single 1.5
CPDs per nucleus were calculated. MED solar-simulated UV-radiation 共UVR兲. Since the neck is
From an area of 200⫻ 200 ␮m2, only within an area of a body site that is naturally heavily sun exposed, it is assumed
160⫻ 80 ␮m2 for Fig. 3共c兲 and 80⫻ 40 ␮m2 for Fig. 3共d兲 that the detected CPDs were induced by solar irradiation. Al-
were CPD-labeled nuclei found, whereas after 1.5 MED irra- though CPDs can be repaired by epidermal cells very
diation, the complete viable epidermis was highlighted. efficiently,28–32 it has been observed that even 10 days after
The amount of CPD-related fluorescence per nucleus irradiation, about 2% of epidermal cells still contain CPDs.33
found after UV-irradiation varied highly between different in- In all the investigated neck samples here, heavily damaged
dividuals, both for solar UV and for fs-irradiation. However, cells in the basal layer of the epidermis can be observed 共Fig.
for fs-irradiation, the variation was smaller. In Table 1, the 3共a兲兴. These CPD-retaining basal cells 共CRBCs兲 occur in hu-
average for all eight volunteers is given, together with its man skin depending on the amount of sun exposure. Some
standard deviation, which indicates the described variations. evidence exists that they may represent epidermal stem
According to the mean values shown in Table 1, the cells,34 which are thought to be the most important targets for
second-highest CPD-related fluorescence was found in neck cutaneous cell aberrations, possibly leading to skin
tissue irradiated with the fs-laser. As described, CPDs were carcinogenesis.35

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 Fischer et al.: Risk estimation of skin damage due to ultra-short pulsed, focused near-infrared irradiation…

Fig. 3 Example of an unirradiated neck tissue slice 共a兲 versus unirradiated buttock tissue 共b兲. Both slices were stained against CPDs. Note the
natural amount of CPD in the sun-exposed body area and heavily stained nuclei in the basal membrane 共white arrows兲.

Fig. 4 Examples of 共a兲 not irradiated, 共b兲 UV-irradiated 共1.5 MED兲, and 共c兲 and 共d兲 fs-irradiated buttock tissue slices. In image 共c兲, one area of CPD
fluorescence due to fs-irradiation was found 共red ellipse兲; image 共d兲 shows the second area 共red ellipses兲, found in three samples.

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 Fischer et al.: Risk estimation of skin damage due to ultra-short pulsed, focused near-infrared irradiation…

100
1,5 MED UV, buttock
CPD-related fluorescence per nucleus (relative

90 fs, buttock, area 1

80 fs, neck
70

60
units)

50

40

30

20

10

0
1 2 3 4 5 6 7 8
volunteer #

(a)

area 2

30
CPD-related fluorescence per

25
nucleus (relative units)

Fig. 6 Action spectrum for the development of CPDs 共䉱兲 in the epi-
20
dermis compared to the action spectrum for skin cancer induction
15 共쎲兲. For comparison, the erythema action spectrum of the Commis-
10 sion Internationale de L’Eclairage 共International Commission on Illu-
mination, CIE兲 is included 共dotted line兲. Modified from Ref. 24; addi-
5
tional references can be found there.
0
1 2 3 4 5 6 7 8
volunteer #

(b) mation with the natural amount of DNA damage after sun
exposure and evaluate the amount of remaining CPD based on
data from the literature. The outermost risk of UV irradiation
can lead to three types of skin cancer, basal cell carcinomas
共BCC兲, squamous cell carcinomas 共SCC兲, and malignant
melanoma 共MM兲.36,37 The first two types have a relatively low
lethality. On the other hand, late malignant melanoma stages
lead to the death of the patient in a number of cases, if the
In the fs-irradiated buttock samples of all volunteers, a melanoma is not removed in time. The connection between
region with CPD-related fluorescence of cell nuclei was ob- UV irradiation and the appearance of BCC or SCC is well
served that can be attributed to the 60-mW fs-NIR-irradiation proven epidemiologically. With MM, the connection is likely,
scheme 关see Fig. 5共a兲, fs-buttock, area 1兴. This region has a but not equally well understood. Using animal investigations
maximum area of 2 ⫻ 10−3 mm2 关Fig. 4共c兲兴. Additionally, a for nonmelanoma tumors, an action spectrum has been
second region could be found in three out of eight volunteers defined by de Gruijl and van der Leun.41
关see Fig. 5共b兲, fs-buttock, area 2兴. This region can be attrib- In the UVB and UVA range, this action spectrum follows
uted to the area irradiated by the scheme using the z-scan with the erythema action spectrum of the Commission Internation-
increasing laser power 共regime 4兲. These second CPD- ale de L’Eclairage 共International Commission on Illumination,
containing regions are small, representing 2.5⫻ 10−4 mm2 CIE; see dotted line, Fig. 6兲. Due to the absorption of shorter
关Fig. 4共d兲兴, respectively. Obviously, after 60-mW fs- wavelengths in the skin the radiation is less effective in the
irradiation with a step width of 5 ␮m or after 150 horizontal UVC range, reaching a maximum at a wavelength of
scans with linearly increasing laser power 共2 mW to ca. 290 to 310 nm, as these data include the protecting effect of
35 mW兲 and a step width of 1 micron, the number of two-and the upper skin layers in the estimation of damage induction in
three-photon excitations is sufficient to induce CPDs in deeper epidermis layers 共e.g., basal cells兲.
deeper cell layers. Taking into account that the microscope Since the primary laser irradiation of the MPLSM used has
focus has a z dimension of about 1.5 ␮m, a double exposure a typical wavelength range of 750 to 850 nm, acute direct
occurs in every 1-␮m scan-step of two consecutive scans and DNA damage by NIR radiation can be excluded. Hence, a real
enhances the formation of CPDs locally. On the other hand, consideration of DNA damage has to take into account exci-
due to skin scattering, deeper tissue layers will receive less tation of energy states usually accessible only by UV absorp-
power. Sun-simulator-exposed skin, on the other hand, shows tion but now generated by multiphoton excitation processes in
significant CPD formation in all viable cell layers of the epi- viable skin layers. Further, the natural DNA repair efficiency
dermis and dermis, and in the basal layer not limited to a after a single exposure by the fs-NIR-laser has been
restricted area. estimated.
One has to bear in mind, however, that the action spectra
4.2 Assessment of the Biological Effectiveness of UV for BCC and SCC, as shown in Fig. 6, could be different in
Damage by Fs-NIR-Laser Irradiation the fs-irradiation situation, because they are related to the ef-
For a better assessment of the damage induced by fs-NIR- fect of irradiation dose at the skin surface. For the mutation
laser irradiation, one has to compare the amount of CPD for- induction in mammalian cells not protected by cornified skin

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 Fischer et al.: Risk estimation of skin damage due to ultra-short pulsed, focused near-infrared irradiation…

layers, an action spectrum for cells in culture was published strand breaks, which recently have been shown to be induced
previously.38 This can also be applied mainly to deeper skin by UVA-radiation. Nevertheless, the large amount of CPDs
layers 共e.g., basal layer兲 because they resemble a kind of 3-D produced in the neck area after natural sun exposure is usually
cell culture domain. repaired by the DNA polymerase repair system in a very
In comparison to the BCC/SCC action spectrum, a much efficient way.
higher effectiveness can be observed at 250 to 280 nm in Taking all the findings and considerations together, at our
nonprotected cells. This is important, since in addition to the irradiation parameters, the risk increase by an fs-NIR-
main 425 to 325 nm, two-photon excitation, there could be a irradiation of a defined small skin area can be assumed to be
nonnegligible amount of short-wavelength UV effects around reduced to negligible values. Even several fs-NIR-irradiation
267 nm being produced in deeper skin layers due to three- sessions delivered at different 0.04-mm2 areas will not in-
photon excitation produced by ultrashort focused laser crease the risk of remaining DNA damage in a measurable
irradiation. way.
The fs-NIR-irradiation experiments have proven that the
Acknowledgments
energy density is high enough to lead to CPD formation, but
whether these CPDs were induced by a two-or three-photon We thank S. Jaspers, G. Hüttmann, K. König, R. Wendel, and
process remains open at the moment. R. Wolber for helpful discussions and J. Batzer and
At a minimum, the measurement of the amount of CPDs K. tom Dieck for their introduction to the use of the sun
offers a possibility of a very rough quantitative risk estimation simulator. We thank R. Börger-Hoppe and R. Keck for the
at the level of DNA damage. The parallel exposure to 1.5 excellent technical assistance. The financial support of Beiers-
MED simulated sun radiation can serve as a “biological ref- dorf AG is gratefully acknowledged.
erence” leading to an exposure of 0.6 and 0.45 MED by fs-
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