Metabolic stress has been implicated in many retinal diseases. It is thus valuable to understand how retinas in the hibernating ground squirrel adapt to extreme environments and successfully cope with metabolic stress. Mitochondria are no doubt at the center of this battle. Although photoreceptor mitochondria have long been a subject of interest, owing to the high density in the inner segment, they cannot be well-studied using light microscopy, which lacks sufficient resolution. Using serial block-face scanning electron microscopy (SBEM), we have successfully generated EM data sets, each from a block of retinal tissue (25 x 25 x 25 m) at 7 nm xy resolution and 20-30 nm z resolution. This new technique generated well-aligned, high-resolution image series that allow us to reconstruct every single mitochondrion in individual cone photoreceptors. Thus, we were able to measure surface area and volume for individual mitochondria as well as the total number of mitochondria in a cone photoreceptor. Interestingly, we observed that mitochondria in a cone photoreceptor are neither completely separated from one another in the form of discrete cylindrical structures, nor do they exist as a single, interconnected syncytia. Instead they exist as variably connected units. In addition, these 3D reconstructions allow us to visualize the spatial orientation of mitochondria within a single cone photoreceptor. Individual mitochondria appear to align themselves parallel to the primary axis of the photoreceptor, suggesting that the mitochondria bundle may have a possible light-guiding function. Using the SBEM technique, we also reconstructed cone photoreceptor mitochondria in hibernating tissues. To minimize variation between samples due to different spatial locations in the retina, we took samples from retinas with similar eccentricities in awake and hibernating animals. We observed several structural alterations in cone mitochondria of hibernating tissues. First, there was a decrease in the total volume and surface area of mitochondria within a single cone. Second, the number of mitochondria per cone increased but connections between them decreased. Third, the spatial arrangement of mitochondria in each cone is less well-aligned with the primary axis of the photoreceptor. To our knowledge, this is the first time that every mitochondrion in individual cone photoreceptors has been successfully reconstructed in 3-dimensions. This data provides a solid foundation for a future modeling study that aims to examine a plausible non-metabolic function of the mitochondria in cone photoreceptor a light-guide feature that maximizes photon delivery efficiency. We also identified structural changes of mitochondria in hibernating tissues, which may have significant functional implications that we are proposing to study in the future.
