Intravital imaging of the anterior segment by lightsheet microscopy

Abstract

Purpose : Intravital imaging approaches have emerged as a powerful technique to study cellular behaviors in the natural environment. Various intravital microscopy strategies have been used to characterize cell dynamics in live mice; however, an optical modality for large-scale imaging of ophthalmology with the subcellular resolution is still a great challenge. Here, we demonstrate a new lightsheet microscopy that enables live imaging of the anterior segment in fluorescent transgenic mice. Furthermore, we highlight the imaging of the corneal endothelium whose detailed healing process remains unclear.

Methods : The excitation source, equipped with 488, 561, and 640nm lasers, is collimated and expanded to the desired beam diameter at 1/e² width of 3mm. The expanded laser beam uses an acousto-optic tunable filter to control the exposure time and wavelength selection. Two water-immersion objective lenses, 10x NA0.3 and 16x NA0.8, are used for excitation and detection respectively. During imaging, the customized eye holder is designed to stabilize the eyeball as well as minimize motion artifacts. The large-scale 3-dimensional (3D) images can be stitched by 5 sub-stacks of which dimensions are 400×512×250µm³.

Results : Large-scale 3D images of ocular surfaces, including the full thickness of corneas and limbus, and the intraocular lens can be visualized. We create wounds with the precise, controllable size of 50μmx50μm by multiphoton femtosecond laser ablation in corneal endothelium to characterize the 4-dimensional wound healing dynamics. During homeostatic status, corneal endothelial cells were outlined in hexagonal shape without proliferation. After wounding, cells on the wound edge exhibited a latent period for about 9 hours before they started migrating as a sheet toward the wound center. This migratory phase lasted from 10 hours to about 40 hours after wounding and closed the wound. Large-scale imaging showed that endothelial cells across the entire cornea remained arrested in G1-phase during the entire healing process without division. Without replenishment of new cells, the healed wound had reduced cell density with compensatory cell enlargement.

Conclusions : This study not only broadens the application of lightsheet microscopy on intravital imaging in ophthalmology but also provides key insights into the spatiotemporal cell dynamics of corneal endothelium during wound healing.

This is a 2021 ARVO Annual Meeting abstract.