Method for single illumination source combined optical coherence tomography and fluorescence imaging of fluorescently labeled ocular structures in transgenic mice

Abstract

In vivo imaging permits longitudinal study of ocular disease processes in the same animal over time. Two different in vivo optical imaging modalities - optical coherence tomography (OCT) and fluorescence - provide important structural and cellular data respectively about disease processes. In this Methods in Eye Research article, we describe and demonstrate the combination of these two modalities producing a truly simultaneous OCT and fluorescence imaging system for imaging of fluorescently labeled animal models. This system uses only a single light source to illuminate both modalities, and both share the same field of view. This allows simultaneous acquisition of OCT and fluorescence images, and the benefits of both techniques are realized without incurring increased costs in variability, light exposure, time, and post-processing effort as would occur when the modalities are used separately. We then utilized this system to demonstrate multi-modal imaging in a progression of samples exhibiting both fluorescence and OCT scattering beginning with resolution targets, ex vivo thy1-YFP labeled neurons in mouse eyes, and finally an in vivo longitudinal time course of GFP labeled myeloid cells in a mouse model of ocular allergy.

Keywords: Animal; Cornea; Fluorescence; GFP; OCT; Optical coherence tomography; Retina; YFP.

Figure 1

OCT-fluorescence imaging system schematic. Illumination and backscattered light is in blue. Fluorescence emission is shown in green. Telecentric imaging optics were used for imaging phantoms, ex vivo murine anterior segment and retina, and in vivo murine anterior segment. Posterior Segment imaging optics were used for imaging in vivo murine retina.

Figure 2

OCT summed volume projection (SVP; gray scale) and fluorescence (green) composite images A) 1951 USAF test chart B) Slide mounted 7μm fluorescent beads

Figure 3

OCT-fluorescence imaging of ex vivo thy1-YFP mouse cornea (top) and retina (bottom). Left) single representative OCT B-scan Right) simultaneously acquired fluorescence image showing fluorescently labelled neurons.

Figure 4

Longitudinal in vivo blue OCT-fluorescence imaging of ocular inflammation in CX3CR1-GFP mouse over one week. For each day, a single OCT B-scan from each volume is on the left, and the simultaneously acquired fluorescence image is on the right. Extraneous structure seen on left side of cornea in day 2 is hydrating gel used to protect the mouse cornea during anesthesia and imaging. The green dots are GFP labeled myeloid cells.

Figure 5

In vivo blue OCT-fluorescence imaging of thy1-YFP mouse retina. A) Averaged repeated B-scans of in vivo retina located at the position indicated by the white box in the volume rendering of Fig. 5D. Strong shadows can be seen below blood vessel lumen due to high absorption and scattering by red blood cells at blue wavelengths B) En face SVP from OCT volume C) Corresponding en face fluorescence image D) Volumetric rendering of OCT volume data.