**2.1 Basic experimental setup**

Figure 1 presents one scheme for a basic photoacoustic experimental setup.

Fig. 1. Example of a basic photoacoustic experimental setup (scheme)

The experimental scheme in Figure 1 shows a (typically mechanical) chopper positioned in front of the light source, in order to modulate the radiation that comes into a monochromator (utilized in PA spectrocopy measurements). Light absorbed by the sample generates acoustic waves inside the PA cell; the PA signal is captured by a microphone (inside the PA cell) that sends it to the lock-in amplifier (also connected to the chopper, to receive information on the frequency modulation). The lock-in amplifier is connected to a microcomputer for data acquisition. In vivo, skin measurements are performed with an open-ended PA cell, in which it is the sample itself that closes the chamber.

## **2.2 Measurements as a function of time**

The PA signal depends on the optical and thermal properties of the sample, which may vary with time due to different factors. When a sample undergoes changes in its

relevance in a large number of very different fields, from the polymerization of dental resins (Balderas-Lopez et al*.*, 1999) to photosynthesis studies (Malkin & Puchenkov, 1997; Herbert

The purpose of this chapter is to present applications of the PA technique in skin research, both in the characterization of skin itself and in transdermal drug delivery studies. The basic experimental setup for such studies will be briefly presented, aiming to help those who may be interested in developing similar studies. Emphasis will be done to *in vivo* measurements, because of its importance in this field. Our objective is to show the usefulness of the PA technique in the biomedical field, particularly in skin research; finally, perspectives for

Figure 1 presents one scheme for a basic photoacoustic experimental setup.

source Chopper Monochromator

Fig. 1. Example of a basic photoacoustic experimental setup (scheme)

Data Acquisition System

open-ended PA cell, in which it is the sample itself that closes the chamber.

**2.2 Measurements as a function of time** 

The experimental scheme in Figure 1 shows a (typically mechanical) chopper positioned in front of the light source, in order to modulate the radiation that comes into a monochromator (utilized in PA spectrocopy measurements). Light absorbed by the sample generates acoustic waves inside the PA cell; the PA signal is captured by a microphone (inside the PA cell) that sends it to the lock-in amplifier (also connected to the chopper, to receive information on the frequency modulation). The lock-in amplifier is connected to a microcomputer for data acquisition. In vivo, skin measurements are performed with an

Sample Lock-in Amplifier

(Optical Cable, optional)

PA Cell +

The PA signal depends on the optical and thermal properties of the sample, which may vary with time due to different factors. When a sample undergoes changes in its

et al., 2000).

**1.1 Objectives** 

future work in this field will be presented.

**2. Photoacoustic measurements** 

**2.1 Basic experimental setup** 

Light

composition or structure (as it occurs during the polimerization of a dental resin, for instance), the propagation of heat inside the sample is also modified, thereby altering the PA signal.

We must also mention the possibility of performing photosynthesis studies using PA measurements as a function of time (W.J. Silva et al., 1995). When PA measurements are performed in photosynthesizing samples as plant leaves, the PA signal presents, in addition to the photothermal component, a photobaric component, resulting from the gas exchanges associated to the photosynthesis process (Acosta-Avalos et al., 1996). This allows the study of the so-called photosynthetic induction, that is, the increase of the net photosynthetic rate that occurs when a plant is shifted from darkness to light (Sui et al., 2011).

As stated by Bodzenta et al. (2002) in their work on PA detection of drug diffusion into a membrane, PA measurements give the possibility for investigations in relatively long time periods. This makes the PA technique suitable for the monitoring of dehydration processes (Lopez et al., 2005) and of changes occurring in time in biological tissues such as skin. It is possible to study, for example, the kinetics of transdermal drug delivery through the analysis of PA measurements as a function of time. One example will be presented at the section 4 of the present chapter.

## **2.3 Studies on the modulation frequency: depth profile**

In thermally thick samples (as skin tissue), only the light absorbed within the first thermal diffusion length (μT) of the sample/tissue contributes to the PA signal (Rosencwaig, 1980). As the thermal diffusion length depends on the modulation frequency (f) of the incident light by the relation

$$
\mu\_{\rm T} = \sqrt{\frac{\alpha}{\pi f}} \tag{1}
$$

where α is the thermal diffusivity of the sample, it is possible to perform depth-profile studies, with the evaluation of the penetration depth of a product (or even a microorganism) in tissue. The possibility of performing depth-profile studies is particularly interesting in the characterization of multilayer systems (as skin itself).

The frequency dependence analysis of the PA signal can also be employed in the determination of the thermal properties (thermal diffusivity, thermal effusivity) of a sample or material (Balderas-Lopez & Mandelis, 2001), including biological tissues as porcine skin (Gao et al., 2005; Qiu et al., 2008).

## **2.4 Measurements as a function of the wavelength: Photoacoustic spectroscopy**

Photoacoustic spectroscopy (PAS) is already incorporated to the roll of useful photothermal techniques since the 1980s (Rosencwaig, 1980; Vargas & Miranda, 1988). Besides the possibility of rendering depth-profile analysis in multi-layered samples, PAS presents at least two additional advantages over other spectroscopy techniques: i) as transmitted and reflected light do not interfere in PAS measurements, it is a "more direct" technique, representing a direct measurement of the light absorption by the sample; ii) it allows the study of optically opaque and highly scattering samples (which could not be analyzed by conventional optical spectroscopy).

Photoacoustic Technique Applied to Skin Research:

**3.1 Skin type classification** 

PA measurements.

Characterization of Tissue, Topically Applied Products and Transdermal Drug Delivery 291

In general, biological tissues can be characterized as highly scattering samples; however, this is not a problem for PA mesurements, in which the signal is based in the direct absorption of radiation. As pointed by Cahen and co-workers (1980), "the relative insensitivity to scattered light of the PA signal makes such measurement an attractive way to measure biological samples *in vivo*". These features explain the potential of the PA technique in the study of opaque materials and complex biological systems such as skin. PA measurements can be employed to determine the absorption characteristics of the skin itself or topically applied

Skin diseases can also be studied through PA measurements. In 2010, Swearingen et al. developed a PA methodology to determine the nature of skin lesions (pigmented and vascular) *in vivo*, which is important because misdiagnosis may even lead to cancerous lesions not receiving proper medical care. These authors irradiated skin with two laser wavelengths (422 and 530nm), with the relative response at these two wavelengths (422nm/530nm) indicating whether the lesion is pigmented or vascular, due to the distinct

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

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) –

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

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

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

defining the so-called "skin phototypes" – is based in clinical, subjective analysis.

**3.2 Skin pigmentation analysis employing photoacoustic measurements** 

products, as well as kinetic changes related to transdermal drug delivery.

absorption spectrum of melanin and hemoglobin (Swearingen et al., 2010).

in defining more specific skin treatments according to each skin type.

classification, which would require experimental evaluation.

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

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 (Melo et al., 2011).

## **2.5 Photoacoustic imaging and tomography**

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

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 interaction.

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