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Two Photon Microscopy for Studies of Xenobiotics in Human Skin

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Abstract

For successful uptake and distribution of drags from transdermal formulations, it is important to understand the skin barrier function, Innovative advances in modem microscopy have provided valuable tools to study the interaction between the skin and xenobiotics. Two-photon microscopy (TPM) allows non-invasive visualization of fluorescent compounds in the skin. The advantages of TPM over conventional confocal microscopy are better light penetration into highly scattering and absorbing tissue such as human skin, improved detection efficiency, limited out of focus photobleaching and reduced phototoxic effects.

We present TPM as an alternative non-invasive in vitro method to study chemical penetration enhancement of fluorescent model drags. The permeability of sulforhodamine B (SRB) through human epidermis was measured with vertical diffusion cells. The absorption was visualized using TPM after 24 h passive diffusion. We have evaluated variations in physicochemical parameters controlling dermal drag uptake induced by the penetration enhancer oleic acid according to methods previously described by Yu et al. Optical sectioning by TPM was compared with cryosectioning.

Oleic acid significantly increased penetration of sulforhodamine. TPM images demonstrate a four-fold increase in the partition coefficient. In addition, a six-fold increase in the concentration gradient was found over stratum comeum. Better light penetration and detection efficiency increase maximum imaging depth in TPM compared to conventional confocal microscopy, however loss of signal due to scattering and absorption is still significant and will affect distribution profiles generated by optical sectioning. A true concentration profile cannot be established without better knowledge about signal losses in the skin.

© 2007 SPIE

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