Abstract
Purpose :
The retina has a high mitochondrial density due to elevated energy requirements. In-vivo imaging of mitochondria at the cellular-level has not been possible due to lack of resolution and available transgenic reporters. Here we used mito-mKate2 transgenic mice to perform the first in-vivo imaging of retinal mitochondria at single-cell resolution with adaptive optics scanning light ophthalmoscopy (AOSLO).
Methods :
Mitochondrial distribution throughout the retina was imaged in mito-mKate2 mice (Jax #32188) with a custom-built AOSLO. In 4 mice, mKate2 fluorescence, located in mitochondria, was captured with 561 nm excitation and 630Δ92 nm emission (25-50 µm pinhole, 1.3 - 2.7 Airy disc diameter, ADD). Distinct retinal layers were imaged at eccentricities between 5 – 20° from the optic disc and structures were measured in Imagej. To validate in-vivo findings and provide ground-truth mitochondrial distribution and anatomy, a single ex-vivo retinal whole mount was also imaged with confocal microscopy (Nikon A1). mKate2 fluorescence was captured with excitation/emission wavelengths similar to that of in-vivo fluorescence capture (28 µm pinhole, 1.5 ADD). Z-stacks were acquired with a step size of 0.5 µm ranging from the GCL to the photoreceptor outer segments.
Results :
In-vivo imaging revealed that ganglion cell somas show fluorescence in the cytoplasm but not the nucleus. A bright band was seen at the OPL where a mosaic of small puncta (0.8 ± 0.1 µm, mean ± 1 SD) and larger aggregations (2 ± 0.4 µm) were suggestive of rod spherule and cone pedicle synaptic complexes. The ONL showed low fluorescence with the exception of sparse, bright puncta (2.2 ± 0.6 µm) spread evenly throughout. Dense mitochondrial aggregation was found at the inner segments of photoreceptors where individual cells could be resolved (1.7 ± 0.2 µm). All in vivo findings matched the anatomy of the same structures imaged ex vivo (Fig. 1).
Conclusions :
To the best of our knowledge, we provide the first images of mitochondria at the cellular scale in the living retina. Monitoring the density and distribution of mitochondria in living retinal cells has the potential to reveal key changes in metabolic demand in health and disease.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.