A comparative study of ex vivo skin optical clearing using two-photon microscopy.

Multiphoton tomography (MPT) is a prospective tool for imaging the skin structure. Aiming to increase the probing depth, a comparative ex vivo study of optical clearing of porcine ear skin was performed by using two optical clearing agents (OCAs), i.e., glycerol and iohexol (OmnipaqueTM ) at different concentrations, which exhibit different osmotic properties. The results show that a topical application of glycerol or OmnipaqueTM solutions onto the skin for 60 min significantly improved the depth and contrast of the MPT signals. By utilizing 40%, 60% and 100% glycerol, and 60% and 100% OmnipaqueTM it was demonstrated that both agents improve autofluorescence and SHG (second harmonic generation) signals from the skin. At the applied concentrations and agent time exposure, glycerol is more effective than OmnipaqueTM . However, tissue shrinkage and cell morphology changes were found for highly concentrated glycerol solutions. OmnipaqueTM , on the contrary, increases the safety and has no or minimal tissue shrinkage during the optical clearing process. Moreover OmnipaqueTM allows for robust multimodal optical/X-ray imaging with automatically matched optically cleared and X-ray contrasted tissue volumes. These findings make OmnipaqueTM more prospective than glycerol for some particular application.