Multimodal Multiphoton Imaging of Human Eye Tissues

Abstract

Purpose: : To evaluate three combined modalities of multiphoton microscopy, second-harmonic generation (SHG), third-harmonic generation (THG), and two-photon-excited fluorescence (2PEF) for imaging cornea and trabecular meshwork in human intact eye tissue.

Methods: : A tunable femtosecond laser chain ( = 700-1250 nm) comprising a titanium-sapphire laser oscillator and an optical parametric oscillator was used to produce 2PEF (380-620nm), SHG (/2=430 or 600nm) and THG (/3= 400nm). Eight corneoscleral discs from eye bank and seven fresh corneal buttons obtained after penetrating keratoplasty were examined with water-immersion objectives. Forward and backward signals were detected and compared.

Results: : The three imaging modalities provide complementary information on intact tissue over the entire thickness of the cornea. THG imaging reveals the tissue morphology, including the epithelium structure with sub-cellular resolution. Polarization-resolved THG microscopy reveals stromal birefringent domains. In phenol-stained corneas, THG also reveals the keratocytes network. SHG imaging probes the distribution of stromal collagen lamellas organization. 2PEF imaging reveals the elastic component of the extra-cellular matrix and the distribution of fluorescent organelles (i.e. mitochondria) in stromal and epithelial cells. The trabeculum images show the three-dimensional organisation of the trabecular lamellas. Emission is predominantly forward directed for THG and SHG but in some cases, images can be recorded in the epi-direction.

Conclusions: : The combined imaging modalities of SHG, THG, and 2PEF microscopy are effective methods to evaluate cornea and trabecular meshwork microstructures in situ. This imaging approach should prove particularly appropriate for assessing corneal and glaucoma physiopathology, and might be amenable to in vivo diagnostics.

Keywords: imaging/image analysis: non-clinical • cornea: basic science • imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound)