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 (Omnipaque^TM ) at different concentrations, which exhibit different osmotic properties. The results show that a topical application of glycerol or Omnipaque^TM 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% Omnipaque^TM 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 Omnipaque^TM . However, tissue shrinkage and cell morphology changes were found for highly concentrated glycerol solutions. Omnipaque^TM , on the contrary, increases the safety and has no or minimal tissue shrinkage during the optical clearing process. Moreover Omnipaque^TM allows for robust multimodal optical/X-ray imaging with automatically matched optically cleared and X-ray contrasted tissue volumes. These findings make Omnipaque^TM more prospective than glycerol for some particular application.