Translator Disclaimer
1 July 2008 Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm
Author Affiliations +
New imaging techniques using near-infrared (NIR) femtosecond lasers (fs-lasers) in multiphoton laser scanning microscopy (MPLSM) have great potential for in vivo applications, particularly in 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 UV source (SSU). DNA damage introduced in the epidermis was evaluated using fluorescent antibodies against cyclobutane-pyrimidin-dimers (CPDs) in combination with immunofluorescence image analysis. Four fs-irradiation regimes (at 800-nm wavelength) were evaluated differing in laser power and step width of horizontal scans. Fs-irradiation did not give CPDs at 15-mW or 30-mW irradiation power using 10 horizontal scans every 5 microns. CPDs could be seen at 60-mW laser power and 5-µm step size and at 35-mW using 1-micron step width. Quantitative comparison of SSU-induced CPDs showed that the 60-mW laser irradiation regime is comparable to UV-irradiation, giving 0.6 minimal erythemal dose (MED). The 1-micron irradiation regime was comparable to 0.45 MED. Under these experimental conditions, the risk of DNA damage due to fs-laser irradiation on skin is in the range of natural UV-exposure.



A few decades ago, ultrashort pulsed laser sources, e.g., femtosecond–laser (fs-laser) systems, became available.1, 2 But it was only recently that they found interest in biomedical research.3 They possess potentials for a multitude of new methods and techniques in different fields of biomedical research, like controlled cell damage in the micrometer range,4 nanosurgery,5, 6 multiphoton laser scanning microscopy (MPLSM),2, 4 DNA-protein cross-linking,6, 7 and optical coherence tomography (OCT).8 So far, these techniques were applied in routine use in vivo only for laser surgery of the cornea, although some other in vivo applications seem to be possible and desirable.9

One reason for the delayed development of in vivo equipment and studies could be related to the fact that little is known about the risk of this type of laser application in vivo, although cell damage has been shown.10 Damage thresholds for in vitro applications can be found.11, 12 For example, the laser power leading to cell damage in microscopy applications was found12 to start with irradiances as low as 2mW . The threshold power depends on pulse duration, mean power, and peak power.11

Using ultrashort pulsed, focused laser irradiation in the near-infrared (NIR) wavelength range, it is possible to reach deeper skin layers (e.g., the stratum germinativum) compared to UV.13 Due to the nonlinear effects of ultrashort focused laser irradiation, biologically relevant skin chromophores are 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 (2PA) and three-photon absorption (3PA) allow excitation of higher energy levels in tissue chromophores. For example, 800nm reaches energy states that are usually accessible only by 400-nm (2PA) or 267-nm (3PA) irradiation. Tissue chromophores like flavins, porphyrines, and/or lipoproteins can be excited by two photons. Simultaneous absorption of three 800-nm photons can excite DNA, NADH, collagen, proteins, and other tissue molecules.

Because of the low pulse energy in the nanojoule range and the low average power (milliwatt) of fs-laser irradiation used in biomedical research, especially in MPLSM, all effects on tissue are connected with nonlinear effects, which occur due to the high peak irradiance, which reaches several hundred GWcm2 . Photochemical damaging induced by linear absorption, which can also occur at low irradiances, is not expected to play a role in the wavelength range between 700nm and 1300nm , which is usually used with fs-lasers. With the exception only of melanin, the endogenous chromophores do not absorb in this wavelength range.14, 15, 16, 17 Therefore, in tissue with low melanin content, the risk of damage due to one-photon absorption (1PA) is expected to be neglectably low. Only biostimulation effects of low-irradiance NIR radiation (below 300mWcm2 ) were described.18, 19, 20, 21, 22

Therefore, only two mechanisms are expected to be relevant for tissue damage, photochemical effects caused by multiphoton absorption and optical breakdown. It is well known that blue and UV light irradiation is able to damage DNA and/or other biomolecules, which absorb in this wavelength range. In skin, photochemical damage results in immediate tissue response (e.g., sunburn) and long-term effects like photoaging or even skin cancer.3, 14

Little is known about the action of fs-lasers in skin. Methods based on “biological dosimetry” are the only option to observe biological effects. Some dosimetric methods use the sensitive and quantitative assay of cyclobutane-pyrimidine-dimers (CPDs) in DNA visualized by immuno-fluorescence detection.23 Cell aberrations may result if induced CPDs are not repaired or are misrepaired by the polymerase repair system.24, 25 The fluorescence intensity of the fluorescent-labeled CPD antibodies can be used as a measure for laser-induced CPD formation. We compared CPDs induced by 1.5 minimal erythema dose (MED) of solar UV.


Materials and Methods


Skin Samples

Skin samples were obtained from healthy volunteers (phototypes II to III) after written consent. Each of eight volunteers gave two skin biopsies, 6mm in diameter, one from the neck and one from the buttock. Samples from the neck were included in this study in order to be able to distinguish effects of extended UV irradiation due to sun exposure from fs-NIR-irradiated skin. After excision, the skin samples were cut in half.



Before skin sample excision, the sensitivity of the volunteers to UV-irradiation was quantified. One minimal erythema dose (MED) was evaluated by visual assessment of six spots irradiated using increasing radiation power of the UV source. The erythema was defined according to COLIPA (The European Cosmetic Toiletry and Perfumery Association) as the first perceptible, clearly defined, unambiguous reddening of the skin 24h after irradiation. UV- and fs-irradiations were performed on freshly excised samples (maximum 1h after excision; see Fig. 1 ).

Fig. 1

Sample preparation and irradiation procedure. (a) Biopsy is divided and irradiated using fs-irradiation (left) or UV-irradiation (right). (b) 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.


For the UV-irradiation, the whole surface of one half of the sample was irradiated using a solar simulator (SU 5000, mut GmbH, Hamburg, Germany) at a UV dose according to 1.5 MED. The UV-irradiation took about 15min . After the UV-irradiation, the samples were frozen in liquid nitrogen for further use. A spectrum of the solar simulator emission is shown in Fig. 2 .

Fig. 2

Emission spectrum of the solar simulator SU 5000.


The other half of the skin sample was irradiated using an ultrashort pulsed, focused laser beam of a Mai-Tai laser (Spectra Physics, Darmstadt, Germany) at a wavelength of 800nm , using a repetition rate of 80MHz and a pulse width at sample layer of about 150fs . For scanning and focusing purposes, a MPLSM (DermaInspect, Jenlab, Jena, Germany) equipped with a Zeiss Plan/Neofluar 40× NA 1.3 objective lens (oil immersion) was used. The DermaInspect device contains a laser emitting NIR in the range between 750 and 850nm according to the specification by the manufacturer. The focus of the laser beam was scanned at various depths within the epidermis and papillary dermis of the excised half of the skin sample. Each horizontal scan was 200×200microns wide and took 32s . Automatic horizontal scanning (parallel to the skin surface) using a fixed step width in the depth of the microscope focus was performed. Four laser irradiation regimes were chosen (Fig. 1):

  • 10 horizontal scans at 15mW laser power ( 96Jcm2 peak fluence, 0.64TWcm2 peak irradiance), 5microns in depth apart from each other;

  • 10 horizontal scans at 30mW laser power ( 191Jcm2 peak fluence, 1.28TWcm2 peak irradiance), 5microns in depth apart from each other;

  • 10 horizontal scans at 60mW laser power ( 383Jcm2 peak fluence, 2.55TWcm2 peak irradiance), 5microns in depth apart from each other;

  • 150 horizontal scans at linear increasing laser power with depth ( 2mW to ca. 35mW , 13to223Jcm2 peak fluence, 0.085to1.5TWcm2 peak irradiance), 1micron in depth apart from each other. Power was increased linearly, since image quality was found to be best when linearly increasing the power with depth.

Peak fluence (or peak radiant fluence) is the time integral of the spherical peak irradiance; it combines the two main paramters influencing dosimetry and hence photochemical effects, in our case, the irradiance and the time. The wavelength and the irradiation time were kept constant during experiments in order to investigate the effects of one parameter, the peak irradiance.

The first three regimes were applied around the basal membrane in order to make sure that the most sensitive cells for risk evaluation, the basal layer cells, will receive the laser light. Three different laser powers (15, 30, and 60mW ) were applied, because with tissue imaging, these laser powers had been shown to cover the possible working range when imaging horizontal scans at basal membrane depth. Laser power was measured using a power meter (Ophir Laserstar) with a coverglass and immersion oil in front of the detector. In this depth, at 15mW , imaging starts to be possible. At 30mW , images can be taken with good contrast. At 60mW , first damaging occurs during scanning (fluorescent scars, as described elsewhere4). The fourth regime covered the whole depth from the surface to the papillary dermis using linearly increasing laser power (Fig. 1). The most suitable laser power to achieve best imaging results was used in order to simulate tissue imaging 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 irradiated areas of about 200×200μm . Between irradiated areas, a space of 400-μm unirradiated tissue was left in order to avoid cross talk between irradiated areas due to light scattering in the skin. At each side of the row of the four irradiated areas, the skin was marked with a black spot in order to retrieve the tiny irradiated areas for the subsequent CPD analysis. The whole fs-irradiation took about 15to20min ; samples were frozen in liquid nitrogen at 196°C after fs-irradiation for further analysis.


Cyclobutane-Pyrimidin-Dimer Antibodies and Staining Protocol

Sections ( 7μm thick) were cut from frozen tissue using a cryostat (Microm, Walldorf, Germany) and were sequentially fixed after slide preparation for 5min in methanol and acetone at 20°C . After fixation, the slides were stored at 20°C . For CPD staining, the samples were rehydrated in TKT 100 ( 50mM Tris pH 7.2; 1M KCl, 0.3% Triton X-100) for 60min at room temperature, washed once in phosphate buffered saline (PBS) for 5min and two times for 5min in 2× SSC ( 300mM NaCl, 30mM Na citrate). The sections were denatured in 0.1N NaOH/70% EtOH for 3min ; dehydrated for 1min each in 70%, 90%, and 100% EtOH; air dried; and incubated with Proteinase K ( 10μg per ml; Sigma, Taufkirchen, Germany) at 37°C for 10min . After three washes for 5min each in PBS, they were incubated with 5% goat serum (Dako, Hamburg, Germany) for 30min at room temperature, rewashed three times for 5min in PBS, and incubated overnight at 4°C with monoclonal antibody specific for CPD (Kamiya Biochemical, Seattle, Washington) diluted 1:1000 in PBS. Sections were then washed three times for 5min each in PBS and incubated with goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC; Dianova, Hamburg, Germany) diluted 1:100 in PBS. After three additional 5-min washes in PBS, slides were mounted with “antifade” (2.3% DABCO; Sigma, Taufkirchen, Germany) in 90% glycerol in 20mM Tris, pH 8.0 and covered. Sections were visualized using a CCD camera (Kappa, Gleichen, Germany) coupled to a fluorescence microscope (Leica DM, Leica, Wetzlar, Germany). Fluorescence intensity was quantified using a digital imaging system (OPTIMAS, Bothell, Washington). Under the premise that the images of the different samples were taken under the same camera conditions and a careful handling of the probes to minimize the influence of photobleaching, the fluorescence intensity is a reliable measure for the amount of induced CPDs (e.g., Refs. 26, 27). Comparing the data of laser-induced CPDs with those induced by 1.5 MED solar UV radiation (UVR), the method can be used to establish the possibility for estimation of skin cancer risks according to current models.



First, an astonishing by high amount of CPD-related fluorescence could be found in unirradiated neck tissue, indicating a recent UV exposure of volunteers. An example using green fluorescent CPD antibodies is shown in Fig. 3a . For comparison, a CPD-stained buttock tissue slice is shown in Fig. 3b. Apart from the nuclei, the images exhibit a low overall fluorescence in the tissue due to a slight unspecific binding of the antibody. While in the neck skin [Fig. 3a] almost all cell nuclei show green fluorescence, the nuclei of buttock tissue are black [Fig. 3b]. Hence, UV-induced CPDs could already be detected in unirradiated neck tissue (due to recent solar UV exposure), while the buttock skin cells show no damage.

Fig. 3

Example of an unirradiated neck tissue slice (a) versus unirradiated buttock tissue (b). 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 (white arrows).


Due to the high amount of CPDs found in native neck tissue (not irradiated during the experiment protocol), a comparison between unirradiated and irradiated neck tissue in order to quantify changes due to fs-laser irradiation was not possible (data not shown). In order to compare the effect of the different irradiation regimes (unirradiated, UV-irradiated, and fs-laser irradiated) only buttock tissue was evaluated. Examples are shown in Fig. 4 .

Fig. 4

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


In the unirradiated buttock samples of all participating volunteers, no CPD fluorescence could be detected [Fig. 4a]. In contrast, many nuclei of the solar simulator irradiated buttock tissues (UV dose equivalent to 1.5 MED) showed CPD fluorescence with high fluorescence intensity in all volunteers [Fig. 4b]. In the fs-irradiated buttock tissue of all volunteers, spatially confined areas of CPD fluorescence could be found [Figs. 4c and 4d]. Horizontal dimensions of these areas were about 150μm , which is about the field of view of the in vitro multiphoton microscope setup used. With the known location of the irradiation regimes due to the marking used on the skin, it seems safe to assume that this fluorescence originates from the CPDs produced using 60-mW fs-irradiation. Additionally, in three buttock samples, a second area with CPD fluorescence could be found [Fig. 4d]. CPD fluorescence in this area was lower and restricted in the horizontal dimensions to a small area. (Only four nuclei show fluorescence signal.) This fluorescence is assumed to be due to the femtosecond irradiation regime applied at varying power from 2to35mW every 1μm in depth.

A quantitative analysis of the CPD fluorescence found in UV-irradiated and fs-irradiated buttock skin for all eight skin samples is given in Fig. 5 . In order to quantify the induced DNA damage, the fluorescence intensity of up to 40 nuclei per slice was measured [arbitrary units] and average values for CPDs per nucleus were calculated.

Fig. 5



From an area of 200×200μm2 , only within an area of 160×80μm2 for Fig. 3c and 80×40μm2 for Fig. 3d were CPD-labeled nuclei found, whereas after 1.5 MED irradiation, the complete viable epidermis was highlighted.

The amount of CPD-related fluorescence per nucleus found after UV-irradiation varied highly between different individuals, both for solar UV and for fs-irradiation. However, for fs-irradiation, the variation was smaller. In Table 1 , the average for all eight volunteers is given, together with its standard deviation, which indicates the described variations.

Table 1

Average and standard deviation of the relative amount of CPDs per nucleus for UV irradiation (1.5 MED) on buttock skin and fs-irradiation on buttock and neck skin.

TreatmentAveragefluorescenceintensity/nucleus [a.u.]SDfluorescenceintensity/nucleus [a.u.]
1.5 MED-UV58.419.1
Fs (neck)27.97.3
Fs (buttock)24.88.0
Fs (buttock 2)18.12.1

According to the mean values shown in Table 1, the second-highest CPD-related fluorescence was found in neck tissue irradiated with the fs-laser. As described, CPDs were found in unirradiated areas of the neck tissue as well, probably due to recent solar exposure of the volunteer [see Fig. 4a]. The areas where fs-irradiation was applied could not be distinguished from the surrounding unirradiated tissue. Hence, the numbers given in Fig. 4 and Table 1 for fs-irradiated neck tissue may be mainly ascribed to CPDs found in native tissue. The fs-irradiation has little or no additional effects in this case. Neck tissue values are given here only for comparison in order to be able to classify sun-exposed tissue in terms of CPD induction.

Buttock skin irradiated at 15-mW and 30-mW fs-laser light using 10 horizontal scans around the basal membrane did not show any CPDs. At 60-mW fs-irradiation, 42% of the CPDs observed in 1.5 MED UV-irradiated buttock skin were found (Table 1). Thus, the amount of CPDs induced by 60-mW fs-irradiation is equivalent to a dose of about 0.6 MED for that small irradiated area.

Irradiation regime four (2to35mW) resulted in 30% CPDs compared to the 1.5 MED UV irradiation. Therefore, this irradiation regime gave 0.45 MED. The fs-irradiated area was much smaller than the UV-irradiated area, resulting in much smaller amounts of fs-irradiated cells.




UV- and Fs-NIR-Irradiation Effects in Human Skin

In the unirradiated areas of fs-exposed samples and untreated buttock samples, no CPD-related fluorescence could be observed [Figs. 4a and 4c]. In contrast, unirradiated areas of fs-exposed neck samples showed already a high amount of CPDs, corresponding to 48% of CPDs induced by a single 1.5 MED solar-simulated UV-radiation (UVR). Since the neck is a body site that is naturally heavily sun exposed, it is assumed that the detected CPDs were induced by solar irradiation. Although CPDs can be repaired by epidermal cells very efficiently,28, 29, 30, 31, 32 it has been observed that even 10days after irradiation, about 2% of epidermal cells still contain CPDs.33 In all the investigated neck samples here, heavily damaged cells in the basal layer of the epidermis can be observed (Fig. 3a]. These CPD-retaining basal cells (CRBCs) occur in human skin depending on the amount of sun exposure. Some evidence exists that they may represent epidermal stem cells,34 which are thought to be the most important targets for cutaneous cell aberrations, possibly leading to skin carcinogenesis.35

In the fs-irradiated buttock samples of all volunteers, a region with CPD-related fluorescence of cell nuclei was observed that can be attributed to the 60-mW fs-NIR-irradiation scheme [see Fig. 5a, fs-buttock, area 1]. This region has a maximum area of 2×103mm2 [Fig. 4c]. Additionally, a second region could be found in three out of eight volunteers [see Fig. 5b, fs-buttock, area 2]. This region can be attributed to the area irradiated by the scheme using the z-scan with increasing laser power (regime 4). These second CPD-containing regions are small, representing 2.5×104mm2 [Fig. 4d], respectively. Obviously, after 60-mW fs-irradiation with a step width of 5μm or after 150 horizontal scans with linearly increasing laser power ( 2mW to ca. 35mW ) and a step width of 1 micron, the number of two-and three-photon excitations is sufficient to induce CPDs in deeper cell layers. Taking into account that the microscope focus has a z dimension of about 1.5μm , a double exposure occurs in every 1-μm scan-step of two consecutive scans and enhances the formation of CPDs locally. On the other hand, due to skin scattering, deeper tissue layers will receive less power. Sun-simulator-exposed skin, on the other hand, shows significant CPD formation in all viable cell layers of the epidermis and dermis, and in the basal layer not limited to a restricted area.


Assessment of the Biological Effectiveness of UV Damage by Fs-NIR-Laser Irradiation

For a better assessment of the damage induced by fs-NIR-laser irradiation, one has to compare the amount of CPD formation 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 (BCC), squamous cell carcinomas (SCC), and malignant melanoma (MM).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 melanoma is not removed in time. The connection between UV irradiation and the appearance of BCC or SCC is well proven epidemiologically. With MM, the connection is likely, but not equally well understood. Using animal investigations for nonmelanoma tumors, an action spectrum has been defined by de Gruijl and van der Leun.41

In the UVB and UVA range, this action spectrum follows the erythema action spectrum of the Commission Internationale de L’Eclairage (International Commission on Illumination, CIE; see dotted line, Fig. 6 ). Due to the absorption of shorter wavelengths in the skin the radiation is less effective in the UVC range, reaching a maximum at a wavelength of 290to310nm , as these data include the protecting effect of the upper skin layers in the estimation of damage induction in deeper epidermis layers (e.g., basal cells).

Fig. 6

Action spectrum for the development of CPDs (▲) in the epidermis compared to the action spectrum for skin cancer induction (●). For comparison, the erythema action spectrum of the Commission Internationale de L’Eclairage (International Commission on Illumination, CIE) is included (dotted line). Modified from Ref. 24; additional references can be found there.


Since the primary laser irradiation of the MPLSM used has a typical wavelength range of 750to850nm , acute direct DNA damage by NIR radiation can be excluded. Hence, a real consideration of DNA damage has to take into account excitation of energy states usually accessible only by UV absorption but now generated by multiphoton excitation processes in viable skin layers. Further, the natural DNA repair efficiency after a single exposure by the fs-NIR-laser has been estimated.

One has to bear in mind, however, that the action spectra for BCC and SCC, as shown in Fig. 6, could be different in the fs-irradiation situation, because they are related to the effect of irradiation dose at the skin surface. For the mutation induction in mammalian cells not protected by cornified skin layers, an action spectrum for cells in culture was published previously.38 This can also be applied mainly to deeper skin layers (e.g., basal layer) because they resemble a kind of 3-D cell culture domain.

In comparison to the BCC/SCC action spectrum, a much higher effectiveness can be observed at 250to280nm in nonprotected cells. This is important, since in addition to the main 425to325nm , two-photon excitation, there could be a nonnegligible amount of short-wavelength UV effects around 267nm being produced in deeper skin layers due to three-photon excitation produced by ultrashort focused laser irradiation.

The fs-NIR-irradiation experiments have proven that the energy density is high enough to lead to CPD formation, but whether these CPDs were induced by a two-or three-photon process remains open at the moment.

At a minimum, the measurement of the amount of CPDs offers a possibility of a very rough quantitative risk estimation at the level of DNA damage. The parallel exposure to 1.5 MED simulated sun radiation can serve as a “biological reference” leading to an exposure of 0.6 and 0.45 MED by fs-NIR-irradiation regimes 3 and 4, respectively. Hence, it is not necessary to measure the local photon dose in deeper skin layers. For BCC and SCC, a risk estimation was performed.39 With a yearly UV dose of 100 MED over 30years at ages from 15 to 45, the risk increases until age 75 by a factor 4. Assuming a linear dose relationship—as is usual in this kind of considerations39—in a rough estimation, one can conclude that one additional exposure by fs-NIR-irradiation regime 3 or 4 increases the risk locally by 0.6% or 0.45%, respectively.

These estimations do not include the fact that only very small, defined areas are irradiated (e.g., in our case, 200×200μm=0.04mm2 ) by MPLSM investigations compared to a sun exposure of total body parts or the whole body. For example, if the face is sun exposed, its area of about 10×10cm (=104mm2) is by a factor 250,000 larger than that exposed during fs-NIR-irradiation.

One has to keep in mind that the risk estimation is based on standard values for a Caucasian skin with phototype II or III and on limited number of ex vivo experiments with a limited number of possible irradiation parameters. Subjects with phototype I are more sensitive to cancer risk due to UV-irradiation and may be more sensitive to fs-irradiation as well. Since the tissue damage is caused by a nonlinear effect, changing laser parameters may result in more severe tissue damage (see Ref. 40).

If the mechanisms of DNA damage differ from that of UV-irradiation, especially when three or more photons are involved that excite to energy states not be reached by UV-irradiation due to the protection of the upper layer of the skin, these experiments may underestimate the cancer risk. In addition, a specially targeted irradiating of cells that are especially important for tumorgenesis (e.g., CRBCs) can increase the tumor risk over a linear extrapolation of the dose or irradiated area.

For future work, it will be interesting to see which role the DNA repair process may play after fs-NIR-irradiation and whether CRBCs can be induced or remain. Furthermore, it is important to investigate alternative UV-induced DNA lesions like 8-oxo-guanine or, even more important, DNA double-strand breaks, which recently have been shown to be induced by UVA-radiation. Nevertheless, the large amount of CPDs produced in the neck area after natural sun exposure is usually repaired by the DNA polymerase repair system in a very efficient way.

Taking all the findings and considerations together, at our irradiation parameters, the risk increase by an fs-NIR-irradiation of a defined small skin area can be assumed to be reduced to negligible values. Even several fs-NIR-irradiation sessions delivered at different 0.04-mm2 areas will not increase the risk of remaining DNA damage in a measurable way.


We thank S. Jaspers, G. Hüttmann, K. König, R. Wendel, and R. Wolber for helpful discussions and J. Batzer and K. tom Dieck for their introduction to the use of the sun simulator. We thank R. Börger-Hoppe and R. Keck for the excellent technical assistance. The financial support of Beiersdorf AG is gratefully acknowledged.



J. A. Valdmanis and R. L. Fork, “Design consideration for a femtosecond pulse laser balancing self phase modulation, group velocity dispersion, saturable absorption, and saturable gain,” IEEE J. Quantum Electron., QE-22 (1), 112 (1986). 0018-9197 Google Scholar


F. W. Wise, I. A. Walmskey, and C. L. Tang, “Simultaneous formation of solitons and dispersive waves in a femtosecond ring dye laser,” Opt. Lett., 13 (2), 129 (1988). 0146-9592 Google Scholar


W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscope,” Science, 248 73 –76 (1990). 0036-8075 Google Scholar


J. A. Galbraith and M. Terasaki, “Controlled damage in thick specimens by multiphoton excitation,” Mol. Biol. Cell, 14 1808 –1817 (2003). 1059-1524 Google Scholar


K. König, I. Riemann, and W. Fritzsche, “Nanodissection of human chromosomes with near-infrared femtosecond laser pulses,” Opt. Lett., 26 (11), 819 –821 (2001). 0146-9592 Google Scholar


S. Lejnine, G. Durfee, M. Murnane, H. C. Kapteyn, V. L. Makarov, and J. P. Langmore, “Crosslinking of proteins to DNA in human nuclei using a 60 femtosecond 266nm laser,” Nucleic Acids Res., 27 (18), 3676 –3684 (1999). 0305-1048 Google Scholar


Ch. Russmann, J. Stollhof, C. Weiss, R. Beigang, and M. Beato, “Two wavelengths femtosecond laser induced DNA-protein crosslinking,” Nucleic Acids Res., 26 (17), 3967 –3970 (1998). 0305-1048 Google Scholar


E. Bordenave, E. Abraham, G. Jonusauskas, N. Tsurumachi, J. Oberle, C. Rulliere, P. E. Minot, M. Lassegues, and B. J. Surleve, “Wide-field optical coherence tomography: imaging of biological tissues,” Appl. Opt., 41 (10), 2059 –2064 (2002). 0003-6935 Google Scholar


B. R. Masters, P. T. C. So, and E. Gratton, “Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin,” Biophys. J., 72 2405 –2412 (1997). 0006-3495 Google Scholar


K. König, H. Liang, M. W. Berns, and B. J. Tromberg, “Cell damage by near-IR beams,” Nature (London), 377 20 –21 (1995). 0028-0836 Google Scholar


K. König, T. W. Becker, P. Fischer, I. Riemann, and K. J. Halbhuber, “Pulse length dependence of cellular response to intense near infrared laser pulses in multiphoton microscopes,” Opt. Lett., 24 (2), 113 –115 (1999). 0146-9592 Google Scholar


K. König, “Cellular response to laser radiation in fluorescence microscopy,” Methods in Cellular Imaging, 236 –251 Oxford University Press, Oxford, England (2001). Google Scholar


A. J. Welch and M. J. C. Van Gemert, Optical-Thermal Response of Laser-Irradiated Tissue, Plenum Press, New York (1995). Google Scholar


J. Longstreth, F. R. de Gruijl, M. L. Kripke, S. Abseck, F. Arnold, H. I. Slaper, G. Velders, Y. Takizawa, and J. C. van der Leun, “Health risks,” J. Photochem. Photobiol., B, 46 20 –39 (1998). 1011-1344 Google Scholar


G. A. Wagnières, W. M. Star, and B. C. Wilson, “In vivo fluorescence spectroscopy and imaging for oncological applications,” Photochem. Photobiol., 68 603 –632 (1998). 0031-8655 Google Scholar


T. Sarna, “Properties and function of the ocular melanin—a photobiophysical view,” J. Photochem. Photobiol., B, 12 215 –258 (1992). 1011-1344 Google Scholar


K. Teuchner, W. Freyer, D. Leupold, A. Volkmer, D. J. Birch, P. Altmeyer, M. Stucker, and K. Hoffmann, “Femtosecond two-photon excited fluorescence of melanin,” Photochem. Photobiol., 70 146 –151 (1999).<0146:FTPEFO>2.3.CO;2 0031-8655 Google Scholar


T. I. Karu, O. A. Tiphlova, Yu. A. Matveyets, A. P. Yartsev, and V. S. Letokhov, “Comparison of the effects of visible femtosecond laser pulses and continuous wave laser radiation of low average intensity on theclonogenicity of Escherichia coli,” J. Photochem. Photobiol., B, 10 339 –344 (1991). 1011-1344 Google Scholar


T. I. Karu, L. V. Pyatibrat, G. S. Kalendo, and R. O. Esenaliev, “Effects of monochromatic low-intensity light and laser irradiation on adhesion of HeLa cells in vitro,” Lasers Surg. Med., 18 171 –177 (1996). 0196-8092 Google Scholar


T. I. Karu, L. V. Pyatibrat, and T. P. Ryabykh, “Nonmonotonic behavior of the dose dependence of the radiation effect on cells in vitro exposed to pulsed laser radiation at lambda=820nm,” Lasers Surg. Med., 21 485 –492 (1997). 0196-8092 Google Scholar


T. I. Karu, T. P. Riabykh, T. A. Sidorova, and Ia. V. Dobrynin, “Comparison of blast cell sensitivity to low-intensity laser radiation and chemotherapeutic drugs,” Dokl. Akad. Nauk, 353 114 –117 (1997). 0869-5652 Google Scholar


N. K. Smol’yaninova, T. I. Karu, G. E. Fedoseeva, and A. V. Zelenin, “Effects of He-Ne laser irradiation on chromatin properties and synthesis of nucleic acids in human peripheral blood lymphocytes,” Biomed. Sci., 2 121 –126 (1991). 0955-9701 Google Scholar


D. L. Mitchell, B. Volkmer, E. W. Breitbart, M. Byrom, M. G. Lowery, and R. Greinert, “Identification of a non-dividing subpopulation of mouse and human epidermal cells exhibiting high levels of persistent UV photodamage,” J. Invest. Dermatol., 117 590 –595 (2001). 0022-202X Google Scholar


R. Greinert, E. W. Breitbart, and B. Volkmer, “UV-radiation biology as part of cancer research,” Life Sciences and Radiation, 139 –155 Springer, Berlin (2004). Google Scholar


A. Van Hoffen, J. Venema, R. Meschini, A. A. van Zeeland, and L. H. Mullenders, “Transcription-coupled repair removes both cyclobutane pyrimidine dimers and 6-4 photoproducts with equal efficiency and in a sequential way from transcribed DNA in xeroderma pigmentosum group C fibroblasts,” EMBO J., 14 360 –367 (1995). 0261-4189 Google Scholar


J. M. Sheehan, N. Cragg, C. A. Chadwick, C. S. Potten, and A. R. Young, “Repeated ultraviolet exposure affords the same protection against DNA photodamage and erythema in human skin types II and IV but is associated with faster DNA repair in skin type IV,” J. Invest. Dermatol., 118 825 –829 (2002). 0022-202X Google Scholar


T. Tadokoro, N. Kobayashi, B. Z. Zmudzka, S. Ito, K. Wakamatsu, Y. Yamaguchi, K. S. Korossy, S. A. Miller, J. Z. Beer, and V. J. Hearing, “UV-induced DNA damage and melanin content in human skin differing in racial/ethnic origin,” FASEB J., 17 1177 –1179 (2003). 0892-6638 Google Scholar


V. J. Bykov, J. M. Sheehan, K. Hemminki, and R. Young, “In situ repair of cyclobutane pyrimidine dimers and 6-4 photoproducts in human skin exposed to solar simulating radiation,” J. Invest. Dermatol., 112 326 –331 (1999). 0022-202X Google Scholar


A. R. Young, C. A. Chadwick, G. I. Harrison, J. L. M. Hawk, O. Nikaido, and C. Potten, “The in situ repair kinetics of epidermal thymine dimers and 6–4 photoproducts in human skin types I and II,” J. Invest. Dermatol., 106 1307 –1313 (1996). 0022-202X Google Scholar


R. J. W. Berg, S. C. de Bueger, K. Guikers, H. van Weelden, W. A. van Vloten, J. C. van der Leun, and F. de Gruijl, “Induction and disappearance of thymine dimers in human skin exposed to UVB radiation: flow cytometric measurements in replicating and nonreplicating epidermal cells,” Photochem. Photobiol., 62 (2), 970 –975 (1995). 0031-8655 Google Scholar


R. N. Freeman, “Variations in excision repair of UVB-induced pyrimidine dimers in DNA of human skin in situ,” J. Invest. Dermatol., 90 814 –817 (1985). 0022-202X Google Scholar


G. Eggset, G. Volden, and H. Krokan, “UV-induced DNA damage and its repair in human skin in vivo by sensitive immunhistochemical methods,” Carcinogenesis, 4 745 –750 (1983). 0143-3334 Google Scholar


S. K. Katiyar, M. S. Matusi, and H. Mukhtar, “Kinetics of UV light-induced cyclobutane pyrimidine dimers in human skin in vivo: an immunhistochemical analysis of both epidermis and dermis,” Photochem. Photobiol., 72 788 –793 (2000). 0031-8655 Google Scholar


B. Volkmer, D. L. Mitchell, E. W. Breitbart, R. Greinert, “Induction of persistent heavily damaged basal cells in human epidermis by solar UV-irradiation,” Google Scholar


R. J. Morris,, “Keratinocyte stem cells: targets for cutaneous carcinogens,” J. Clin. Invest., 106 3 –8 (2000). 0021-9738 Google Scholar


V. O. Melnikova and H. Ananthaswamy, “Cellular and molecular events leading to the development of skin cancer,” Mutat Res., 571 91 –106 (2005). 0027-5107 Google Scholar


B. K. Armstrong and A. Kricker, “The epidemiology of UV induced skin cancer,” J. Photochem. Photobiol., B, 63 8 –18 (2001). 1011-1344 Google Scholar


F. Zölzer and J. Kiefer, “Wavelength dependence of inactivation and mutation induction to 6-thioguanine-resistance in V79 Chinese Hamster fibroblasts,” Photochem. Photobiol., 40 49 –53 (1984). 0031-8655 Google Scholar


H. Slaper and J. C. van der Leun, “Human exposure to ultraviolet radiation: quantitative modelling of skin cancer incidence,” Human Exposure to Ultraviolet Radiation, 155 –171 Elsevier Science Pub. Co., Inc., New York (1987). Google Scholar


A. Vogel, J. Noack, G. Hüttmann, and G. Paltauf, “Mechanisms of femtosecond laser nanosurgery of cells and tissues,” Appl. Phys. B, 81 1015 –1047 (2005). 0946-2171 Google Scholar


R. J. W. Berg, F. R. de Gruijl, and J. C. van der Leun, “Interaction between ultraviolet A and ultraviolet B radiations in skin cancer induction in hairless mice,” Cancer Res., 53 4212 –4217 (1993). 0008-5472 Google Scholar
©(2008) Society of Photo-Optical Instrumentation Engineers (SPIE)
Frank F. Fischer, Beate Volkmer, Stefan Puschmann, Ruediger Greinert, Wolfgang Breitbart, J. Kiefer, and R. Wepf "Risk estimation of skin damage due to ultrashort pulsed, focused near-infrared laser irradiation at 800 nm," Journal of Biomedical Optics 13(4), 041320 (1 July 2008).
Published: 1 July 2008

Back to Top