**3.1 Skin type classification**

290 Acoustic Waves – From Microdevices to Helioseismology

In PAS measurements, the emission spectra of the light source is typically obtained through measurements using black carbon powder (or other black material) as the sample, with all

PAS can also be employed in skin research. In 2004, Benamar and co-workers presented a PAS study on the effect of dihydroxyacetone, frequently employed for artificial tan. Measurements were carried out in the presence and absence of dimethylisosorbide (a solvent for dihydroxyacetone), on excised human skin. By monitoring the PAS signal intensity with time in the UV (300-400nm) range, these authors demonstrated that dihydroxyacetone in combination with dimethylisosorbide enhances the process of tanning (Benamar et al., 2004). Recently, Melo et al. (2011) applied PAS to evaluate the penetration rate of *Helicteres gardneriana* extract, topically applied for anti-inflammatory purposes. Experiments were conducted *ex vivo* in mice. Croton oil was applied into both mouse's right and left auricles to induce inflammatory response, and the left auricle was treated with the extract. The strong anti-inflammatory effect observed for the *Helicteres gardneriana* extract was associated with the deep percutaneous penetration observed for the formulation, according to PA data

Photoacoustic imaging is based on the production of acoustic waves following irradiation by a short pulse of light whose absorption generates local heating and transient thermoelastic expansion (Balogun et al*.*, 2009). According to Beard (2009), haemoglobin "represents the most important source of endogenous contrast" in PA imaging. This makes the technique particularly indicated to studying tissue abnormalities as tumors and other diseases related

Recently, Hu and Wang (2010) presented "PA tomography" as a method combining high spatial resolution and optical absorption contrast, important in microvascular imaging and characterization. Reviewing the "major embodiments of PA tomography" (microscopy, computed tomography and endoscopy), they have analyzed the methods employed in different studies, including hemodynamic monitoring, determination of hemoglobin concentration, evaluation of oxygen saturation, studies of blood flow and tumor-vascular

Besides being applied to soft tissues, PA imaging can also be employed to hard tissues. Li and Dewhurst (2010) have applied a PA imaging system with a near-infrared (NIR) pulsed laser to obtain images from both soft tissue and post-mortem dental samples. They have also performed simulations (based on the thermoelastic effect) to predict initial temperature and pressure fields within a tooth sample, observing that values are maintained below the corresponding safety limits. In this way, the results presented by Li and Dewhurst show

Biological materials are sometimes difficult to study employing conventional techniques that require previous preparation of the samples, because these materials can have its properties significantly altered by preparation processes as solubilization, for example. The PA technique does not require previous preparation; it can be described as a non-invasive

that the PA technique can be sucessfully applied to image both soft and hard tissues.

**3. Photoacoustic measurements and the characterization of skin** 

technique that allows even *in vivo* measurements.

to changes in the structure and oxygenation status of the vasculature (Beard, 2009).

the remaining measurements being normalized with respect to the lamp spectrum.

(Melo et al., 2011).

interaction.

**2.5 Photoacoustic imaging and tomography** 

Skin type classification is important not only for medical or clinical purposes, but also for pharmaceutical and cosmetic industries, following the idea that an objective, precise characterization of skin could be useful in the design of new topically applied products and in defining more specific skin treatments according to each skin type.

However, in dermatology, there is still no universal agreement about the best method for classifying skin, as even the widely accepted method proposed by Fitzpatrick (1988) – defining the so-called "skin phototypes" – is based in clinical, subjective analysis.

More recently, Baumann (2006a, 2006b) proposed a new skin type classification, according to which 16 different skin types are defined from the combination of four parameters, as skin can be characterized as: i) pigmented or nonpigmented; ii) dry or oily; iii) sensitive or resistant; and iv) wrinkled or tight. Baumann´s skin typing is based on an extensive research, performed with 1400 volunteers. However, it relies essentially on the response of volunteers to a questionnaire; therefore, it does not fulfill "per se" the need of an objective classification, which would require experimental evaluation.

PA measurements have a potentially important role to play in an experimental approach to skin type classification. In 2000, Schmidt and co-workers conducted non-contacting, *in vivo* PAS measurements in skin (performed in 50 volunteers), in the VIS-NIR range, seeking an objective determination of pigmentation, blood microcirculation and water content of human skin (Schmidt et al., 2000). According to these authors, strong spectral variations observed within the same skin type are probably based on the natural variability of human skin and in the subjective clinical evaluation of the skin type; nevertheless, PAS results obtained show good correlation between PA data and (clinically evaluated) skin type, indicating that skin type determination could indeed be performed through the analysis of PA measurements.

## **3.2 Skin pigmentation analysis employing photoacoustic measurements**

In 2004, Viator and co-workers proposed a method for the determination of the epidermal melanin content employing a PA probe using a Nd:YAG (neodymium, yttrium, aluminum, garnet) laser at 532nm (Viator et al*.*, 2004). Ten human subjects with skin phototypes I–VI

Photoacoustic Technique Applied to Skin Research:

was gently pressed against the opposite face.

**3.2.2 Results and discussion** 

Results are presented in Table 1.

(comparison among groups: p=0.009, ANOVA)

of sun exposure.

the thickness of the skin layer under study is about 30µm.

FASBio/UNIVAP, with a volunteer positioned for measurement.

and this classification was compared to the phototype classification.

Characterization of Tissue, Topically Applied Products and Transdermal Drug Delivery 293

17Hz. The electret microphone structure was described by Marquezini et al*.* (1990). The PA cell has a cylindrical body and two opposite, parallel faces (one is closed by a thin glass layer and the other, by the sample itself). For the modulation frequency employed (17Hz),

PA measurements were recorded as a function of time (200 readings for each measurement, in 0.5s intervals, up to a total of 100s per measurement). During measurements, one face of the PA cell was closed with a thin transparent window, while the forearm of the volunteer

Figure 2 shows the PA experimental setup employed for in vivo skin measurements at the

Measurements were performed in 57 female volunteers, between 20 and 30 years-old. Initially, each volunteer answered a questionnaire according to their daily routine associated to skin care; volunteers were also clinically evaluated and, as a result, they were classified

Before measurements, the skin area to be evaluated was cleaned with cotton embedded in alcohol 70%. The PA signal was then recorded for the inner and outer faces of both forearms. Volunteers were then classified according to the respective PA signal amplitude,

Initially, a comparison between the PA signal amplitude of the inner and outer faces was performed, showing a highly significant statistical difference (paired t-test, p<0,005), with higher PA amplitude being observed for the outer face of the forearm. This result can be attributed to the higher pigmentation level of the skin region continuously exposed to solar radiation, demonstrating that skin constitution and aspect are clearly influenced by the level

After clinical evaluation of the volunteers for skin phototype (following Fitzpatrick classification), PA results were grouped according to the phototype of each volunteer.

> **Skin phototype (Fitzpatrick) PA signal amplitude (mV)**  II 1.26 ± 0.05 a III 1.59 ± 0.09 ab IV 1.70 ± 0.10 ab V 1.80 ± 0.10 b

Table 1. PA signal amplitude (mV) for the inner face of the forearm, for each skin phototype (average ± standard error). Different indexes (a, b) indicate significant statistical difference

Comparison among phototype groups was performed and significant statistical difference was verified (as we can see in Table 1), showing that the PA signal level (amplitude) for the

Afterwards, the PA signal amplitude for each volunteer (average values for the inner face of the forearm) allowed the division of the volunteers in two groups, "pigmented" (P) and "non-pigmented" (NP), following the Baumann proposal. As the average PA signal

inner forearm tends to scale with skin phototype, as defined by Fitzpatrick.

according to skin phototype, following Fitzpatrick classification (Fitzpatrick, 1988).

were tested using the PA probe and visible reflectance spectroscopy (VRS); melanin content was evaluated through each of these methods, and a good linear fit (r2=0.85) was obtained for the plot of PA x VRS.

Pigmentation skin level can also be evaluated through simple, direct PA measurements employing non-laser light sources. Actually, PA measurements have been performed at the Laboratory of Photoacoustic Technique Applied to Biological Systems (FASBio), at UNIVAP (Brazil). The objective of such *in vivo* measurements was to classify different skin types according to the amplitude of the PA signal, which can be associated to the corresponding pigmentation level of the skin.

In the following subsections, we present this straightforward PA approach to skin characterization according to the level of pigmentation, employing PA measurements in volunteers. Experimental results are compared both to Fitzpatrick and Baumann clinical skin type evaluations.
