Multiphoton Imaging for Visualization and Ablation of the Trabecular Meshwork - A New Approach to Glaucoma Surgery

Abstract

Purpose: : Currently, filtering surgery is limited by scarring and trabecular surgery which should allow for reduction of outflow resistance is lacking appropriate instrumentation. Multiphoton imaging combines diagnosis on a cellular level with the option of ablation in the micron range. We explored the potential application of multiphoton imaging for visualization and simultaneous ablation of trabecular tissue for non-invasive glaucoma surgery.

Methods: : A compact solid-state mode-locked 90 MHz Ti: sapphire femtosecond laser with a wide wavelength range 715-930 nm and 140 fs pulse duration, connected to a modified multiphoton laser scanning microscope Zeiss Axiovert 510-Meta was used to visualize and ablate trabecular meshwork and anterior segment tissues in anaesthetized mice and rabbits. Autofluorescent cellular structures were imaged at 750 nm incident wavelength, whereas extracellular collagen fibers were visualized at 850 nm. The images were collected in both tangential and sagittal levels using an 20 x 0.9 objective at 2 µm z-steps. For intratissue ablation, the mean laser power was enhanced to about 210 mW. After imaging and ablation, animals were killed and the eyes were enucleated for histological analysis.

Results: : Optical sections were obtained from corneal, scleral, and chamber angle tissues. Multiphoton imaging provided high-resolution images of cornea, limbus and sclera at cellular level. The trabecular meshwork (TM) was identified through the sclera, avoiding opening of the eye. After femtosecond laser power enhancement, highly precise intratissue ablation within the trabecular meshwork was performed. Histology confirmed not only the nature of the cellular images obtained by multiphoton imaging, but also the absence of detectable collateral damage adjacent to the ablation in the TM.

Conclusions: : In vivo multiphoton-autofluorescence imaging and ablation by femtosecond laser pulses within TM is possible ab externo, and may prove to be a clinically applicable novel option for non-destructive trabecular surgery.

Keywords: laser • microscopy: light/fluorescence/immunohistochemistry • outflow: trabecular meshwork