In vivo imaging of the inner retinal layer structure in mice after eye-opening using visible-light optical coherence tomography

Abstract

The growth of the mouse eye and retina after birth is a dynamic, highly regulated process. In this study, we applied visible-light optical coherence tomography (vis-OCT), a non-invasive imaging technique, to examine developing retinal layer structures after eye-opening. We introduced a resampled circumpapillary B-scan averaging technique to improve the inter-layer contrast, enabling retinal layer thickness measurements as early as postnatal day 13 (P13) - right after eye-opening. We confirmed vis-OCT measurements using ex vivo confocal microscopy of retinal sections at different ages. Our results demonstrate that vis-OCT can visualize the developmental murine retinal layer structure in vivo, which offers us new opportunities to better characterize the pathological alterations in mouse models of developmental eye diseases.

Keywords: In vivo imaging; Optical coherence tomography; Retinal development.

Figure 1.

The retina undergoes dynamic changes as the animal continues to grow during postnatal development. (A) Confocal microscopy images of retinal sections at P10, P30, and P60 immunostained by DAPI for nuclei. GCL: ganglion cell layer; IPL: inner plexiform layer; INL: inner nuclear layer; OPL: outer plexiform layer; (B) Eyeball diameter change; (C) Flat-mount retina diameter change; (D) Total retina thickness change. *: P<0.05; **: P<0.01; and ***: P<0.001.

Figure 2.

Resampled circumpapillary averaging. (A) Example en face image. The rings highlight resampling paths. (B) Magnified view of the resampling path enclosed by the green box on ring 1 in panel A; (C) Resampled circumpapillary averaged B-scan imaged along the path highlighted by ring 2 in panel A; (D) Magnified view of the inner retina of the area highlighted by the purple box in panel C with overlaid resampled averaged A-line from the center of panel D shown in red. Scale bar: 50 μm.

Figure 3.

Comparing retinal thickness measurements from direct and resampled circumpapillary averaging at P14 and P60. (A) B-scan images using direct averaging. Scale bar: 100 μm.; (B) B-scan images using resampled circumpapillary averaging. Scale bar: 100 μm; (C) Comparing the coefficients of variation of retinal thickness measurements at P14; (D) Comparing the coefficients of variation of retinal thickness measurements at P60.

Figure 4.

Measured retinal layer thickness changes. (A-D) Representative vis-OCT en face images (top), non-averaged B-scan images (middle), and resampled circumpapillary averaged B-scan images (bottom) from P14, P20, P30, and P60. The red lines in the en face images highlight the location of the non-averaged B-scan images. The yellow curves in the en face images and the yellow boxes in the non-averaged B-scan images correspond to the locations of the resampled circumpapillary averaged B-scan images. Blue asterisks mark the location of hyaloid shadows. Scale bar: 200 μm; (E) En face acutance values from different age groups; (F) Intralayer contrast due to hyaloid shadows measured from the non-averaged B-scan images at different retinal layers from different age groups; (G-J) Measured TRT, INL, IPL, and RNFL/GCL thickness change with age from resampled circumpapillary averaged B-scan images.

Figure 5.

Validating in vivo vis-OCT measurements with ex vivo confocal microscopy measurements. (A) In vivo vis-OCT direct averaged B-scan image from P60; (B) Ex-vivo confocal microscopy image from P60; (C) Correlation constant values between mean vis-OCT and mean confocal microscopy measurements of different retinal layers from different age groups. (D) Correlating vis-OCT and confocal microscopy measured retinal layer thicknesses.