Abstract
Purpose: Applications of advanced imaging techniques for the characterization of retinal pathology in small animal models could provide insight into structural and molecular changes as a function of injury or disease progression. Integration of the volumetric data obtained in vivo by SD-OCT with ex vivo autofluorescence using multiphoton-microscopy (MPM) in the murine retina can be used to investigate the temporal characteristics of retinal injury and disease.
Methods: In vivo imaging of retinal tissue was obtained using SD-OCT. Controlled photothermal injury was induced to investigate the cellular and molecular changes associated with the retinal response to laser radiation. MPM studies were performed in fresh and fixed retinal tissue using a Prairie Technologies MPM with illumination wavelengths between 720 and 1020nm acquired at 20 nm intervals for autofluorescence imaging. Additional contrast agents were used to preferentially stain photoreceptors for MPM imaging.
Results: Distinct retinal features were observed throughout the entire depth of the tissue and characterized across the range of wavelengths, allowing for the determination of several structures and layers. Using a combination of these techniques, clearly definable structures included several functional retinal layers (GCL, IPL, INL, OPL, ONL, PRL), major vasculature, the photoreceptor mosaic, and adherent RPE cells. Contrast provided by Mitotracker staining of the rod outer-segments provided high-resolution visualization of the photothermal lesion boundaries in the photoreceptor mosaic.
Conclusions: Depth-resolved imaging of scattered light using OCT and the retinal autofluorescence using MPM can both provide complementary cellular and molecular views within the distinct layers of the retina.