Sensorless Adaptive Optics for Two Photon Excited Fluorescence Imaging of the Mouse Retina

Abstract

Purpose : Visualization of the Retina Pigmented Epithelium (RPE) is important for studying Age-Related Macular Degeneration (AMD) and other vision related diseases. However, imaging the RPE can be challenging particularly in mouse models of human diseases. Our purpose is to non-invasively image the RPE cell mosaic in mice through the combination of Two Photon Excited Fluorescence (TPEF), Optical Coherence Tomography (OCT), and Sensorless Adaptive Optics (SAO).

Methods : In this work, we developed a SAO OCT imaging system with simultaneous TPEF. A tunable femtosecond pulsed laser was used to investigate TPEF intensity versus wavelength, and also permitted OCT imaging with the same light source. Two deformable elements were used for aberration correction, including a Variable Focus Lens (VFL; defocus) and a deformable mirror. We implemented image-based SAO, which corrected aberrations up to the 21^st Zernike mode. The image-based SAO could be performed using either the depth-specific OCT images or the TPEF images.

Results : We imaged B6(A)-Rpe65^rd12/J (RPE65) transgenic mice at ~18 months of age. The OCT was used to align the mouse to the system, and to provide cross sectional images to guide the VFL in adjusting the focal plane to the retinal layer interest. Figure 1(a) shows an OCT B-scan focused on the Nerve Fiber Layer (NFL) with a ~570 µm FOV. The FOV was reduced to ~350 µm for SAO, and the en face OCT before and after aberration correction are shown in Figure 1(b). The VFL was used to shift the focal plane to the RPE. Figure 2 shows TPEF images that were acquired after SAO using a center wavelength of 760 nm, 780 nm, 800 nm, and 820 nm.

Conclusions : We have shown the ability to perform SAO in vivo in the mouse eye, as indicated by the improvement in the OCT image quality in Figure 1. We have also imaged the intrinsic fluorescence from the RPE with SAO TPEF in the RPE65 mouse at different excitation wavelengths. Our results show a stronger TPEF signal that more clearly delineates the RPE mosaic at 760 nm as compared to 820 nm.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

Fig. 1. (a) OCT B-scan with the focal plane at the NFL. (b) En face OCT images before and after SAO. Scale bars: 50 µm.

 

Fig. 2. TPEF images with the focal plane at the RPE using different central wavelengths for fluorescence excitation. The red arrows point at the same RPE cell across each image. Scale bar: 50 µm.