Two-photon noninvasive imaging of murine retina in vivo

Abstract

 

Purpose

 

Two-photon excited fluorescence (TPF) imaging of retina of living animals can help investigating mechanisms of retinal diseases and the development of ophthalmic therapies. Previously reported systems for two-photon imaging in living animal eye required application of a custom contact lens. Here we report the optical design of a periscope for coupling a light beam into the mouse eye and capturing the emitted TPF that provides subcellular images of the retinal pigmented epithelium (RPE) without a custom contact lens.

 

Methods

 

The optical design of this periscope maximizes on mouse eye numerical aperture and provides an interface with the TPF imaging system without the need for a custom contact lens. Images of RPE and retina were obtained from albino Rpe65^−/− and wild type (WT) mice with a TPF system equipped with the new periscope, a laser delivering 75 fs pulses, a dispersion compensation unit and sensor-less adaptive optics. Before imaging mice were anesthetized by injection of a solution consisting of ketamine and xylazine in distilled water. All animal procedures and experiments were approved by the Institutional Animal Care and Use Committee at Case Western Reserve University and conformed to recommendations of both the American Veterinary Medical Association Panel on Euthanasia and the Association for Research in Vision and Ophthalmology.

 

Results

 

The periscope imaged the RPE with 730 nm excitation light. RPE images obtained with the periscope revealed crisp and clear structure of RPE mosaic - Fig. 1. The minimal mean power of the beam required to obtain a TPF image was 5mW. The images have wider field of view, better defined RPE mosaic and the fluorescence spectrum exhibited less noise than those previously reported with a contact lens.

 

Conclusions

 

This newly designed periscope is capable of obtaining high quality TPF images of the RPE and retina in live mice without using custom contact lenses. This new periscope provides an interface with a commercial microscope system and could easily be modified to image the retina and RPE in the eyes of different animal species.  

 

TPF imaging of RPE in living WT and RPE65^-/^- mice. A: mouse eye illuminated with the newly designed periscope; A1: image of the RPE in an RPE65^-/^- albino mouse; A2, A3: images of the RPE of the WT albino mouse. Excitation wavelength: 730 nm. Mean power at cornea: 20mW-A1 and 25mW-A2,A3. Scale bars: 50um-A1,A3 and 100um-A2.