A Custom Multiphoton Microscopy Platform for Live Imaging of Mouse Cornea and Conjunctiva

Summary

Presented here is a multiphoton microscopic platform for live mouse ocular surface imaging. Fluorescent transgenic mouse enables the visualization of cell nuclei, cell membranes, nerve fibers and capillaries within the ocular surface. Non-linear second harmonic generation signals derived from collagenous structures provide label-free imaging for stromal architectures.

Abstract

Conventional histological analysis and cell culture systems are insufficient to simulate in vivo physiological and pathological dynamics completely. Multiphoton microscopy (MPM) has become one of the most popular imaging modalities for biomedical study at cellular levels in vivo, advantages include high resolution, deep tissue penetration and minimal phototoxicity. We have designed an MPM imaging platform with a customized mouse eye holder and a stereotaxic stage for imaging ocular surface in vivo. Dual fluorescent protein reporter mouse enables visualization of cell nuclei, cell membranes, nerve fibers, and capillaries within the ocular surface. In addition to multiphoton fluorescence signals, acquiring second harmonic generation (SHG) simultaneously allows for the characterization of collagenous stromal architecture. This platform can be employed for intravital imaging with accurate positioning across the entire ocular surface, including cornea and conjunctiva.

Introduction

The ocular surface structures, including the cornea and conjunctiva, protect other deeper ocular tissues from external disturbances. The cornea, the transparent front part of the eye, functions both as a refractive lens for directing light into the eye and as a protective barrier. Corneal epithelium is the outermost layer of the cornea and consists of distinct layers of superficial cells, wing cells and basal cells. Corneal stroma is composed of sophisticatedly packed collagenous lamellae embedded with keratocytes. Corneal endothelium, a single layer of flat hexagonal cells, has an important role in maintaining the transparency of cornea by keeping corneal stroma in a....

Protocol

All animal experiments were conducted in accordance with procedures approved by the Institutional Animal Care and Use Committee (IACUC) of the National Taiwan University and Chang Gung Memorial Hospital.

1. Multiphoton microscopy setup

1. Build a system based on an upright microscope with water immersion 20x 1.00 NA objective (Figure 1A).
2. Use Ti: Sapphire laser (with tunable wavelength) as the excitation source. Set the laser output wavelength at 880 nm for EGFP and 940 nm for tdTomato (Figure 1A).
3. Include two dichroic mirrors (495 nm and 580 nm) for th....

Results

Using this live imaging platform, the mouse ocular surface can be visualized at cellular levels. To visualize individual single cells in the ocular surface, we employed the dual fluorescent transgenic mice with EGFP expressed in the nucleus and tdTomato expressed in the cell membrane. The collagen-rich corneal stroma was highlighted by SHG signals.

In corneal epithelium, superficial cells, wing cells and basal cells (Figure 2) were visualized. In the dual fluoresc.......

Discussion

This custom-built MPM imaging platform with a control software was used for intravital imaging of mouse epithelial organs, including skin¹⁰, hair follicle¹⁰ and ocular surface^9,10 (Figure 1A). The custom-built system was used for its flexibility in changing the optical components for various experiments, since the beginning of our project. This imaging methodology is versatile for comme.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AVIZO Lite software	Thermo Fisher Scientific		Version: 2019.3.0
Bandpass filters	Semrock	FF01-434/17 FF01-500/24 FF01-585/40
Dichroic mirrors	Semrock	FF495-Di01-25x36 FF580-Di01-25x36
Galvano	Thorlabs	GVS002
Jade BIO control software	SouthPort Corporation	Jade BIO
Oxybuprocaine hydrochloride	Sigma	O0270000
PMT	Hamamatsu	H7422A-40
Polyesthylene Tube	BECTON DICKINSON	427401
Stereotaxic mouse holder	Step Technology Co.,Ltd	000111
Ti: Sapphire laser	Spectra-Physics	Mai-Tai DeepSee
Upright microscopy	Olympus	BX51WI
Vidisic Gel	Dr. Gerhard Mann Chem-pharm. Fabrik GmbH	D13581
Zoletil	Virbac	VR-2831