Label- free SHG imaging and spectral FLIM of corneas using a sub-15 fs laser microscope

Alterations to the corneal cell metabolism or to the structural organization of collagen fibrils occur in several corneal and systemic pathologies. In this work we resort to multiphoton microscopy corneal imaging to achieve a characterization of the corneal state. Using fluorescence lifetime imaging microscopy (FLIM) the assessment of the metabolic state of corneal cells is possible, whereas second harmonic generation (SHG) imaging can be used to assess corneal structural alterations. A sub-15 fs near-infrared laser source with a broad excitation spectrum was used for SHG imaging and FLIM. The broad spectrum allows simultaneous excitation of both metabolic co-factors. The signals were collected by a photomultiplier tubes (PMT) detector with 16 simultaneous recording channels, which allowed the separation of fluorophores autofluorescence based on their emission wavelengths. We were able to successfully image ex-vivo human and porcine cornea at multiple depths. Simultaneous NADH and flavin autofluorescence, SHG of collagen fibrils, and stroma autofluorescence imaging was performed which may in future allow an improved characterization of the metabolic and structural alterations of the corneal tissue due to pathophysiological conditions. This would be an important step towards a better understanding of corneal dystrophies and systemic metabolic disorders.