Abstract
Purpose :
Versatile techniques based upon adaptive optics (AO) ophthalmoscopy have allowed for multimodal visualization of the live human retina but replicating findings with high-resolution microscopic imaging analogs is still difficult. In order to cross validate in vivo findings from an AO ophthalmic imaging system, a customized microscope system was added, enabling the examination of subcellular features in fixed or ex vivo ocular tissue.
Methods :
A custom-built AO instrument was modified to integrate a modular microscope assembly (Thorlabs Cerna). Utilization of a 40X water-dipping objective (Nikon N40X-NIR) allows for enhanced resolution and light collection efficiency compared to in vivo ophthalmic imaging. Fluorescent beads (Phosphorex) were used to empirically characterize the point spread function and resolution of the imaging device. Fixed retinal tissue from macaque and human donors was immersed in PBS and immobilized with a slice anchor (Warner Instruments) for imaging, and the resulting images were compared to in vivo results from healthy subjects.
Results :
This addition of a microscope assembly enables sample visualization utilizing the existing multimodal AO system, allowing for concurrent imaging of confocal reflectance, nonconfocal split detection, and fluorescent channels. The apparent full width at half maximum intensity of 200 nm fluorescent beads was 770 nm ± 14 nm, demonstrating the capability of resolving subcellular features. The increased lateral resolution, along with confocal optical sectioning allows for visualization of subcellular features in fixed or ex vivo ocular tissue. Specifically, confocal reflectance and split detection imaging of the photoreceptor layers of a macaque retina shows sub-micron scaled patterning within individual cone photoreceptor cells. While images obtained from healthy human eyes with in vivo AO ophthalmic imaging suggest the existence of similar subcellular features, these features are below the resolution capability of the current AO ophthalmoscope.
Conclusions :
Validating findings of cellular damage and subcellular features from AO ophthalmic imaging is difficult absent histological confirmation. Our strategy combines the advantages of a multimodal AO ophthalmic imaging system with the superior resolution of microscope optics allowing more rigorous correlation of in vivo findings.
This is a 2020 ARVO Annual Meeting abstract.