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Application of a Dermatological Laser Scanning Confocal Microscope for Investigation in Skin Physiology
J. Lademann, H. Richter, N. Otberg, F. Lawrenz, U. Blume-Peytavi, W. Sterry
ABSTRACT
The dermatological laser scanning confocal microscope, Optiscan Stratum, was applied to in-vivo investigations in skin physiology. The radiation of an Ar+ laser at 488 nm was used to excite the topically applied food dyes, curcumin and sodium fluorescine. The radiation was transferred by an optical fiber to a handpiece which was set directly onto the skin surface.
INTRODUCTION
The action and protection properties of topically applied substances and cosmetic drugs are significantly influenced by their penetration and distribution characteristics. Precise knowledge and specific investigation of penetration pathways of topically applied substances is highly important for the development of molecules and drugs with optimal penetration capacities. The investigation of the penetration pathways and the homogeneity of the distribution of substances in the skin, requires the application of time and space resolved measurements. An optimal method would be a technique enabling non-invasive, real time measurements related to the upper part of the human skin, the stratum corneum, as this skin layer presents the barrier for the penetration of external substances into the human body. This layer of skin has a thickness between 10 - 100 µm depending on the body site. The stratum corneum possesses reservoir properties for topically applied substances, so that the penetration kinetics, the so-called dermatopharmacokinetics, are determined by this upper skin layer and by the properties of the applied substances.
Online methods based on optical and spectroscopic measurements using the absorption or scattering properties of topically applied substances are well suited for penetration and distribution studies. The most promising methods are the attenuated total-reflection spectroscopy (ATR) [1], opto-acoustic spectroscopy (OAS) [2], optical coherence tomography (OCT) [3, 4] and the laser scanning confocal microscopy (LSCM) [5, 6].
Laser scanning confocal microscopy has become a very promising method of measurement in skin physiology after it became possible to investigate any site of the body in-vivo. For these measurements, the object lens and the scanning module of the microscope are incorporated in a hand piece, which can be applied directly onto the skin surface.
In the present paper, the application of a dermatological laser scanning confocal microscope for penetration and distribution measurements of topically applied substances is reported.
MATERIAL AND METHODS
The experiments were carried out using the dermatological laser scanning confocal microscope 'Stratum', Optiscan Ltd., Melbourne. The radiation of an Ar+ laser at 488 nm was used to excite the topically applied fluorescent dye curcumin and sodium fluorescine. The radiation was transferred by an optical fiber to the probe which contained the scanning system. The investigated field of vision is 200 x 200 µm. The probe scans in real time, scan frequency depends on scan resolution. For maximum resolution the frame rate is one frame per second. The optical window of the handpiece was set directly onto the skin surface. The fluorescence emission was collected by the objective lens and transferred by the optical fiber to a photo detector. The depth scan was realized by changing the focus position manually, by using the imaging depth control on the handpiece.
The investigations were carried out on healthy male and female volunteers (age: 25 - 45 years). The food dyes, curcumin and sodium fluorescine, were applied in ethanol or in a sunscreen emulsion (2 mg/cm2). The concentration of the dyes in the formulations was 1%. The treated skin area was 4 x 5 cm2. Penetration and distribution measurements were carried out at different times after topical application.
RESULTS AND DISCUSSION
Analysis of the structure of the skin layers
After the application of sodium fluorescine, the different layers of the human skin can be well distinguished by changing the depth of the focal plane in the tissue. Different skin layers are presented in figure 1. The corneocytes of the stratum corneum appeared primarily on the skin surface. The stratum granulosum, the stratum spinosum and subsequently the stratum basale, can be distinguished by scanning the laser focus deeper into the skin. In deeper skin layers (200 µm), the papillary dermis becomes visible just below the basal layer. This depth corresponds to the maximum penetration depth of laser radiation (λ 488 nm) into the tissue.
Changes in the structure of different skin layers caused by diseases can be well distinguished by LSCM measurements.
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| Stratum corneum | Stratum granumosum | ||
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| Stratum spinosum | Papillary dermis | ||
| Fig. 1: Detection of substances in different layers of the living human skin by laser scanning confocal microscopy measurements | |||
Analysis of the distribution of topically applied substances in the stratum corneum
A variety of cosmetic products and pharmacological agents are intended either to stay on the skin surface or to penetrate into certain layers of the human skin. Sunscreens are one typical application, where the product should stay on the surface and exert protective properties.
A typical result is presented in figure 2. The dye-containing emulsion is distributed non-homogeneously in the stratum corneum and partly located in the furrows of the skin (arrow). After improving the homogeneity of the sunscreen distribution, by optimization of the formulation, the sun protection factor (SPF) can be significantly increased without changing the UV-filter concentration [7]. In this way, LSCM measurements are an easy method to investigate the properties of formulation concerning their distribution in the human skin.
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| Fig. 2: Distribution of topically applied substances in the stratum corneum | |||
Investigation of the penetration kinetics of topically applied substances into the stratum corneum
The penetration kinetics of a dye-containing formulation were investigated with LSCM by analyzing the penetration depth at the same skin area at different time points after application. The fluorescence images of the stratum corneum of the forearm of a volunteer, 5 min and 20 min after application of a curcumin-containing emulsion, is presented in figure 3. The curcumin dye is only located in the first layers of the stratum corneum 5 min after application. Later, deeper corneocyte layers could also be detected step by step, because the dye had reached these layers as a result of the penetration process. After 20 min, 3 layers of the stratum corneum became visible (left picture of figure 3). The shifted structure of the stratum corneum can be clearly distinguished.
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| Fig. 3: Deeper cell layers of the stratum corneum become visible during penetration of the fluorescent dye curcumin into the horny layer (left picture - 5 min after application; right picture - 20 min after application) | |||
Penetration pathways
In figure 3, we have demonstrated how the topically applied dye-containing formulation penetrated deeper into the stratum corneum. The highest fluorescence intensity was always detected in the lipid layers that surround the corneocytes. This demonstrates that the lipid layers of the stratum corneum are an efficient penetration pathway for topically applied substances, as has been discussed in the literature [8,9,10]. The influence of the follicular penetration on the total penetration process has been underestimated in past years. This is the reason why the follicle orifices present only 0.1 percent of the total skin surface. In contrast, it was demonstrated that even relatively large particles, such as titanium dioxide (Ø 100 µm), used in sunscreens, can penetrate into the hair follicles [11]. Additionally, it was shown that active and passive follicles can be distinguished concerning the penetration of topically applied substances [12].
Laser scanning confocal microscopy allows the online investigation of the follicular penetration process in-vivo. Penetration kinetics into the follicles can be detected by applying a fluorescent dye onto the skin. In figure 4, this penetration process is demonstrated by analyzing the distribution of the fluorescent dye at different time points after application. On the images, it can clearly be seen, how the substance has penetrated into the follicles.
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| Fig. 4: Penetration of the fluorescent dye curcumin into a hair follicle | |||
This method is well-suited to analyze the follicular penetration in-vivo and to investigate the phenomena of active and passive follicles, concerning the penetration of topically applied substances. However, these investigations are not limited to hair follicles. The penetration of substances into the sweat glands could also be analyzed.
First investigations demonstrated that in the case of sweat glands, it is also possible to distinguish between active and passive glands during the penetration process. This effect is demonstrated in figure 5. The left picture shows an active sweat gland into which the dye-containing emulsion has penetrated. A passive sweat gland can be seen on the right image. No dye could be detected inside this sweat gland. Both types of sweat glands were detected closely in the same skin area.
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| Fig. 5: Detection of sweat glands, where the topically applied curcumin dye was penetrated (active) and non penetrated (passive). | |||
SUMMARY
In-vivo LSCM measurements are well-suited to investigate the skin structure and penetration processes of topically applied substances in real time. This non-invasive method presents an efficient tool for clinical diagnostics and therapy control in dermatology. By analyzing the distribution of topically applied substances, LSCM measurements can be used to optimize the efficiency and skin physiological parameters of topically applied drugs and cosmetic products.
LITERATURE