Contrast enhancement in combined two-photon second harmonic imaging of skin by using hyperosmotic agents

We used combined simultaneous two-photon excitation fluorescence microscopy (TPE) and second harmonic generation microscopy (SHG) on human skin tissue slices. We studied the effect caused by topical application of optical clearing agents (OCAs). We demonstrated that hyperosmotic agents as glycerol, propylene glycol and glucose in aqueous solution, are all effective in improving excitation light penetration depth and in enhancing image contrast. The effect caused on acquired images by sample immersion in OCAs or in their aqueous dilution, was studied. We observed a similar clearing effect with TPE and SHG acquisitions, with different effectiveness and rising time for each agent. The TPE acquired data are in good agreement with a simple diffusion model developed. From the SHG acquisition some different behaviour was observed. All three agents are potentially bio-compatible and effective in reducing scattering, improving light penetration depth and image contrast. Use of OCA can be suitable for in vivo application in two-photon microscopy, as well as in other techniques performing optical biopsy of human skin tissue.