Adaptive-Optics SLO imaging combined with phase-variance OCT for precise 3D localization of fluorescent cells in the mouse retina

Abstract

 

Purpose

 

To evaluate feasibility of a mouse Adaptive Optics - Scanning Light Ophthalmoscope (AO-SLO) and phase variance Optical Coherence Tomography (pv-OCT) for precise 3D localization of cells in the mouse retina in vivo.

 

Methods

 

A mouse AO-SLO system has been used to investigate with high resolution (~1 µm) and small FOV (~150 x 150 µm) retinal vasculature and morphology of fluorescent cells in mice. In parallel the retinas of the same mice were imaged with custom pv-OCT systems, allowing precise non-invasive mapping of volumetric retinal vasculature at ~4µm resolution over a large FOV (~1.6 x 1.6mm). The AO-SLO uses custom 0 Dpt. contact lens mounted in the entrance pupil plane of the system to allow precise mouse positioning and long time imaging. Several pigmented Wild Type (c57BL/6) mice as well as mice with GFP labeled microglia (Cx3cr1^GFP/+), and neutrophils (129Sv lys-EGFP) were used as the imaging targets.

 

Results

 

The two imaging techniques offer complementary views of mouse retinal morphology on macro (pv-OCT) and micro (AO-SLO) scales. The figure shows mouse vasculature imaged with pv-OCT (C,D - label free) and AO-SLO (E - Fluorescein Angiography) in Wild Type (c57BL/6) mouse.<br /> The retinal vasculature visualized with both pv-OCT and AO-SLO acts as the anchor between two modalities, allowing easy registration and precise localization of cellular structures imaged by AO-SLO to specific retinal layers. The pv-OCT allowed label-free mapping of retinal microvasculature that was used as a reference map for imaging areas of interest with AO-SLO.

 

Conclusions

 

Application of two distinct retinal imaging modalities enables precise 3D localization of AO-SLO fluorescent sources in the retina in vivo. This capability is especially valuable for longitudinal studies of the morphology, transformations and migration of fluorescently tagged cells in vivo  

 

Pv-OCT and AO-SLO fluorescein angiography (FA) from one mouse. A. En face intensity projection image of an OCT volume. B. B-scan from the same volume through optic disc (red arrow in A). C. En face pv-OCT of vascular beds. D. Depth color-coded visualization of high resolution pv-OCT (red box in C). E. Six images from AO-SLO z-stack from retinal region outlined in blue in C spanning from NFL (upper left) to just “below” the OPL (lower right).