Determination of porcine corneal layers with high spatial resolution by simultaneous second and third harmonic generation microscopy.

We describe a dual, second harmonic generation (SHG) and third harmonic generation (THG) microscope, with the aim to obtain large-scale images of the cornea that can simultaneously resolve the micron-thick thin layers. We use an Ytterbium femtosecond laser as the laser source, the longer wavelength of which reduces scattering and allows simultaneous SHG and THG imaging. We measure one-dimensional SHG and THG profiles across the entire thickness of pig cornea, detected in both the forward and backward directions. These profiles allow us to clearly distinguish all the porcine corneal layers (epithelium, stroma, Descemet's membrane and endothelium). From these profiles, longitudinal cross sectional images of the corneal layers are generated, providing large scale topographic information with high-spatial resolution. The ability to obtain both SHG and THG signals in epi-detection on fresh eyes gives promising hopes for in vivo applications.