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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

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 a.......

Representative 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 surface9,10 (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.......

Acknowledgements

We thank the grant support from Ministry of Science and Technology, Taiwan (106-2627-M-002-034, 107-2314-B-182A-089, 108-2628-B-002-023, 108-2628-B-002-023), National Taiwan University Hospital (NTUH108-T17) and Chang Gung Memorial Hospital, Taiwan (CMRPG3G1621, CMRPG3G1622, CMRPG3G1623).

....

Materials

NameCompanyCatalog NumberComments
AVIZO Lite softwareThermo Fisher ScientificVersion: 2019.3.0
Bandpass filtersSemrockFF01-434/17
FF01-500/24
FF01-585/40
Dichroic mirrorsSemrockFF495-Di01-25x36 FF580-Di01-25x36
GalvanoThorlabsGVS002
Jade BIO control softwareSouthPort CorporationJade BIO
Oxybuprocaine hydrochlorideSigmaO0270000
PMTHamamatsuH7422A-40
Polyesthylene TubeBECTON DICKINSON427401
Stereotaxic mouse holderStep Technology Co.,Ltd000111
Ti: Sapphire laserSpectra-PhysicsMai-Tai DeepSee
Upright microscopyOlympusBX51WI
Vidisic GelDr. Gerhard Mann Chem-pharm. Fabrik GmbHD13581
ZoletilVirbacVR-2831

References

  1. DelMonte, D. W., Kim, T. Anatomy and physiology of the cornea. Journal of Cataract & Refractive Surgery. 37 (3), 588-598 (2011).
  2. Van Buskirk, E. M. The anatomy of the limbus. Eye (London). 3, 101-108 (1989).
  3. Hodges, R. R., Dartt, D. A.....

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Multiphoton MicroscopyLive ImagingMouse CorneaMouse ConjunctivaDual Fluorescent Transgenic MouseEGFPTdTomatoSHGStereotaxic Mouse HolderEyelid RetractionEye GelZ stack Imaging

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