2.1.

Volunteers

The optical properties of human skin in vivo were determined on the volar and dorsal aspect of the forearm as well as on the thenar in a subject study ( $n=20$ , 14 women, 6 men) using UV optoacoustics. The study design was approved by the local ethics committees, and all volunteers gave informed consent.

2.2.

Phototype/MED

Subjects with different phototypes (I: $n=3$ , II: $n=7$ , III: $n=5$ , IV: $n=5$ ) were tested for their individual minimal erythema dose (MED) at the volar side of the forearm. The subject’s individual MED at the volar side of the forearm was determined by chromameter measurements (Minolta CR200, Minolta, Osaka, Japan) according to COLIPA recommendations, defining the erythemal threshold as a change in $a^{*}$ of $+2.5$ units $24\mathrm{h}$ after irradiation with a solar simulator (M.U.T. GmbH, Hamburg, Germany). For subjects with Fitzpatrick phototype characteristics in-between two types, the phototype classification was decided with the help of their MED at the volar side of the forearm, resulting in a corresponding MED range for each photoype: phototype I $200–230\mathrm{mJ}∕{\mathrm{cm}}^2$ , phototype II ca. $250\mathrm{mJ}∕{\mathrm{cm}}^2$ , phototype III $260–280\mathrm{mJ}∕{\mathrm{cm}}^2$ , and phototype IV $300–400\mathrm{mJ}∕{\mathrm{cm}}^2$ .

2.3.

Optoacoustics

Optoacoustic measurements were carried out in reflection mode. Laser radiation is delivered via a UV-enhanced optical fiber that illuminates a ca. $4{\mathrm{mm}}^2$ large area of the skin through UV-transparent ultrasound gel, which also ensures acoustic coupling between the sample and a poly(vinylidene fluoride) ultrasound detector above the skin. More details of the optoacoustic sensor have been described elsewhere.^{15, 16}

A pulsed UV laser system ( $\mathrm{NL}303\mathrm{G}+\mathrm{PG}122∕\mathrm{UV}$ , Ekspla, Lithuania) provided wavelength tunable radiation in the range from $290\text{to}341\mathrm{nm}$ with pulse duration of $5\mathrm{ns}$ and pulse energies in the range of $200\text{to}700\mu \mathrm{J}$ . The energy of each pulse was recorded by an energy monitor at the UV fiber. For one optoacoustic wavelength scan, the subject’s skin was exposed to an erythemally weighted UV dose on the order of $50\mathrm{mJ}∕{\mathrm{cm}}^2$ .

Within this wavelength range, optoacoustic measurements were carried out in $3\text{-}\mathrm{nm}$ steps at the volar and dorsal aspect of the forearm as well as on the thenar. Three spectra were recorded for averaging within a circular area of $<2.5{\mathrm{cm}}^2$ on the relevant skin areas of each subject. On the volar aspect of the forearm, these measurements were carried out on both arms and again $72\mathrm{h}$ after the first measurements on the right arm (no indication of significant differences between these three measurement triples). If necessary, the skin area was shaved at the dorsal aspect of the forearm to minimize the influence of hair on the optoacoustic measurements. In any case, measurement spots were aimed to be in the hair-free space between follicles.

For signal processing and analysis, we used a self-developed simulation software based on the equations for thermooptical excitation of sound in a dissipative medium with inhomogenous optical properties. The program takes into account the actual geometry of our experimental setup and was calibrated using melanin dyed tissue phantoms,¹⁶ thus also incorporating sound attenuation, influences of the electronic setup, etc. For analysis, the complete optoacoustic signal is simulated. Even though the optical properties of the skin are expected to change considerably with depth—especially from stratum corneum to viable epidermis—we could not resolve a depth dependency in our measured data. Student’s t-test was used for statistical analysis of significant differences between the UV penetration depths at different skin sites.

3.1.

Skin Sites

The skin on the inner and outer sides of the arm is very much alike; it may be a little thicker and more strongly pigmented on the outer side of the arm. Figure 1 shows the average wavelength-dependent penetration depths $(290–341\mathrm{nm})$ at the three sites together with their 90% confidence intervals. As expected, the penetration depths at the volar and dorsal forearm show fairly similar characteristics. Ultraviolet radiation can penetrate deeper into the skin at the volar side throughout the spectrum ( $p<0.05$ for 335–305 and $296\mathrm{nm}$ , $p<0.005$ for 329–311 and $305\mathrm{nm}$ ; one-tailed paired t-test). This is in accordance with the consideration that skin on the dorsal forearm of the same subject should display the same pigmentation as on the volar side, plus possibly some amount of extra facultative pigmentation because the outer side of the forearm is more strongly exposed to the sun than the inner side. Equal or stronger pigmentation should result in equal or shallower penetration depths comparing these two sites for the same subject. Minimal penetration depths of ca. $20\mu \mathrm{m}$ are found at $290\mathrm{nm}$ for both sites. Toward longer wavelengths, the penetration depths are slowly increasing in both spectra (up to $50–60\mu \mathrm{m}$ , see Fig. 1 for details). At the dorsal side, maximal penetration depths are found at $320\mathrm{nm}$ followed by a weak decrease toward $341\mathrm{nm}$ . At the volar side, the penetration depths do not show a maximum but reach a plateau in the UVA (315–499 nm).

Fig. 1

Penetration depths (1/e-level) of UV radiation in human skin. Error bars mark 90% confidence intervals. The wavelength dependence of the penetration depth is similar at the volar (◼) and dorsal (●) aspect of the forearm. It is increasing from $290\mathrm{nm}$ (volar: $20.4\pm 3.5$ $90\%\text{-}\mathrm{CI}\mu \mathrm{m}$ ; dorsal: $18.9\pm 4.8\mu \mathrm{m}$ ) toward longer wavelengths. In the UVA, penetration depths are fairly constant with a maximum of $61.1\pm 5.7\mu \mathrm{m}$ at $329\mathrm{nm}$ at the volar and a maximum of $50.9\pm 7.2\mu \mathrm{m}$ at $341\mathrm{nm}$ at the dorsal aspect. Spectral characteristics are different at the thenar (▲). Here, the penetration depth is only very slowly rising in the UVB ( $10.9\pm 3.5\text{to}19.4\pm 4.1\mu \mathrm{m}$ at $290–314\mathrm{nm}$ ) and increases steeply in the UVA (up to $134.6\pm 18.4\mu \mathrm{m}$ at $341\mathrm{nm}$ ). The bar scheme at the side indicates the ranges of important skin layers in normal skin (not thenar) for orientation (s.c.: stratum corneum).

The spectrum of penetration depths at the thenar differs significantly from those at the forearm (volar/thenar: $p<0.005$ for 341–332 and $323–290\mathrm{nm}$ , one-tailed paired t-test; dorsal/thenar: $p<0.05$ for 341–329 and $320–290\mathrm{nm}$ , $p<0.005$ for 341–332 and $317–299\mathrm{nm}$ , one-tailed paired t-test): whereas in the UVB (290–315 nm) the penetration depth only slowly rises from well above $10\mu \mathrm{m}$ , it increases steeply in the UVA so that radiation even penetrates deeper at the thenar than at the forearm sites at wavelengths longer than ca. $326\mathrm{nm}$ .

As already indicated when comparing volar and dorsal forearm spectra, a one-tailed paired t-test was used because we expect a change of optical properties from skin site to skin site caused by UV adaptation and/or a thicker horny layer to go in the same direction for each subject. Intraindividual comparison of the site spectra supports this assumption. However, even if this is not assumed and a two-sided test is used, the results substantially stay the same: for the difference between volar and dorsal forearm spectra, $p$ is then slightly larger than 0.05 for 335 and $296\mathrm{nm}$ and drops from highly significant $(p<0.005)$ to significant $(p<0.05)$ for $326\mathrm{nm}$ ; no changes in the level of significancy occur for the t-test of volar/thenar, and for dorsal/thenar, $p$ only turns from highly significant to significant at $299\mathrm{nm}$ .

3.2.

Phototypes

Figure 2 takes a closer look at the phototype dependence of the penetration depths at the three sites. This time the average penetration depth was calculated separately for the different phototypes. Of course, the 90% confidence intervals are fairly large here because of the small number of subjects: $n=3$ for phototype I, $n=7$ for phototype II, $n=5$ for phototype III, and $n=5$ for phototype IV. Still, the breakdown of the data into the four phototype groups allows an interesting perspective.

Fig. 2

Average spectra of the penetration depths at different skin sites: (a) volar aspect of the forearm, (b) dorsal aspect of the forearm, and (c) thenar (ball of the thumb). Mean spectra and 90% confidence intervals are shown for phototypes I–IV; phototype I $(n=3)$ : ◼, phototype II $(n=7)$ : ●, phototype III $(n=5)$ : ▲; and phototype IV $(n=5)$ : ▼.

At the volar side of the forearm [Fig. 2a], penetration depths of phototypes I–III are very close in the UVB. Penetration depth is minimal at $290\mathrm{nm}$ $\left(20\mu \mathrm{m}\right)$ and rises constantly up to ca. $55\mu \mathrm{m}$ at $314\mathrm{nm}$ . It is maximal in the range from $317\text{to}329\mathrm{nm}$ and decreases again toward longer wavelengths. In the UVA, the close agreement between the three phototypes is lost to some degree and the curves are less smooth. Penetration depths are now higher (approximately $60–80\mu \mathrm{m}$ ) in phototypes I compared to phototypes II and III (well above $40\mu \mathrm{m}$ to ca. $60\mu \mathrm{m}$ ). Phototype IV behaves a little differently from the other three types. Penetration depth stays at a low level of $<20\mu \mathrm{m}$ from $290\mathrm{nm}$ up to $299\mathrm{nm}$ . Then it rises to another plateau at $60\pm 5\mu \mathrm{m}$ from $332\mathrm{nm}$ onward. This implicates higher penetration depths in phototypes I and IV in the UVA range above ca. $330\mathrm{nm}$ than in phototypes II or III.

At the dorsal side of the forearm [Fig. 2b], phototypes I and II as well as III and IV seem to be grouped throughout the UVB and up to $\sim 320\mathrm{nm}$ . Penetration depth is again rising from shorter to longer wavelengths, and it is higher in the light skin types than in the darker ones. Phototypes behave differently toward longer wavelengths. In type IV, the penetration depth is constantly rising from $\sim 20\mu \mathrm{m}$ at $290\mathrm{nm}$ up to above $45\mu \mathrm{m}$ at $341\mathrm{nm}$ . Types III and II reach a kind of plateau in the UVA. However, this level is reached earlier in type II (at ca. $318\mathrm{nm}$ ) than in type III (at ca. $326\mathrm{nm}$ ) and it is as high as $50\mu \mathrm{m}$ in skin type II and $<45\mu \mathrm{m}$ in type III. In phototype I, penetration depths reach maximal levels of $50–55\mu \mathrm{m}$ in the range of $318–329\mathrm{nm}$ and then decrease toward longer wavelengths down to $40\mu \mathrm{m}$ .

The phototype-dependent penetration depths calculated for the thenar [Fig. 2c] show an interesting trend: the penetration depths in the horny layer are considerably higher in phototype I than in the other skin types throughout the measured spectral range. The confidence intervals of phototype I data are relatively large due to the small number of subjects in this group; thus, this difference is not significant. If, however, the variance in a larger group of phototype I subjects turned out to be equally small, as that of the group containing phototypes II–IV, the difference between the thenar absorption spectra would become significant for wavelengths other than 335–323 and $314\mathrm{nm}$ .