Method for quality-assessing excised human corneal buttons for nanoparticle visualization

This study introduces a methodology for analyzing light transmission and nanoparticle penetration in excised human corneas using multiphoton microscopy. By developing an accessible approach utilizing the laser from a confocal or multiphoton microscope for assessing corneal quality via light transmission, we measured transmission to be over 90% through corneal tissue across a wavelength range of 700-1060 nm. The method enables precise optical measurements crucial for validating tissue quality before further experimentation without the need for separate equipment. Additionally, the study demonstrates the use of multiphoton microscopy with a femtosecond laser to image nanoparticle penetration into the human corneal stroma using an epithelium-off <i>ex vivo</i> model. Fluorescent mPEG-coated nanoparticles were imaged at different depths, co-located with collagen fibers that were imaged with second-harmonic generation. This dual approach, combining optical transparency assessment with nanoparticle tracking, provides a reliable method for studying corneal tissue and evaluating the potential impact of nanoparticle-based therapies.