High‐resolution two‐photon excitation microscopy of ocular tissues in porcine eye

Abstract

Two-photon excitation laser scanning microscopy (TPM), based on nonlinear optical (NLO) response under high irradiance, is currently being extensively employed for diagnostic purposes in biomedical fields and becomes more and more an interesting imaging technique in the intact bulk tissue examination. In this study, this nonlinear-excitation imaging technique including two-photon-mediated autofluorescence (2PF) and second harmonic generation (SHG) was employed to investigate the microstructures in the whole-mount scleral, retinal, and corneal tissues of porcine eyes with intracellular spatial resolution and high signal-to-noise ratio. Image acquisition was based on the intense 80 MHz femtosecond (fs) near-infrared (NIR) laser pulses, emitted from a mode-locked solid-state titanium:sapphire system. By integrating a high-numerical aperture diffraction-limited objective, the whole-mount ocular specimens could be viewed from the surface of eye globes further to a 200 microm depth. Under high light irradiance at the order of MW-GW/cm2, more than one photon was simultaneously absorbed by endogenous molecules in ocular tissues. The cellular and fibrous components of whole-mount scleral and corneal tissues were selectively displayed in situ by in-tandem detection of 2PF and SHG with high efficiency without the assistance of any exogenous dye. NLO images of fibroblasts and mature elastic fibers in sclerae as well as of the retina radial Müller glial cells, ganglion cells, bipolar cells, photoreceptors, and retina pigment epithelial (RPE) cells were acquired with subcellular spatial resolution. In particular, the microstructural topography of cells and extracellular components in the whole-mount ocular tissues was elucidated in situ. The combination of the sensitive image acquisition technique allows to selectively studying of three-dimensional (3-D) architecture of cellular microstructures and extracellular matrix arrangement in situ at substantial depths in bulk tissues. The data obtained provided the primary knowledge for further studies of imaging entire eye globes based on two-photon excitation microscopy.