Diffusion of optical clearing agents in skin studied by two-photon microscopy

We report the effects on two-photon excitation microscopy of applying optical clearing agents to human skin tissue samples. We demonstrate that the agents glycerol, propylene glycol and glucose in aqueous solution are all effective in enhancing penetration depth (by up to a factor of 2) and in increasing image contrast (by up to a factor of 90 at 80 μm depth) in 150 μm thick sections. We analysed the dynamics of the clearing process, by developing a simple theoretical model based on the free diffusion of the agent into the tissue. In experiments employing simultaneous two-photon excitation and second harmonic generation microscopy similar contrast was produced. A preliminary measurement of the clearing effect on a bulk skin sample is also presented. All three agents are potentially biocompatible and effective in reducing scattering; hence, in improving light penetration depth and image contrast. As such, they could be suitable for in vivo application in two-photon microscopy, as well as in other techniques performing optical biopsy of human skin tissue.