Development of Confocal Multispectral Imaging Using a Common Commercial Device and Application in a Healthy Aging Mouse Model

Purpose: To develop implementable confocal multispectral imaging (MSI) by adapting data capture and developing novel analysis on a commercially available device. Using an aging mouse model as proof-of-principle confocal MSI was compared against current reference-standard flood-illuminated hyperspectral imaging (HSI). Methods: Mice aged three, five, and eight months old (n = 14-19/group) underwent confocal scanning laser ophthalmoscopy retinal imaging (MultiColor module on Spectralis OCT) at three laser wavelengths returning: blue (BR, 486 nm), green (GR, 518 nm), and infrared reflectance (IR, 815 nm). Images were focused in 2D steps over a range of 20D. Average retinal reflectance was calculated for wavelengths across the dioptric range and corrected for chromatic aberration. Flood-illuminated HSI from 320 to 680 nm was also conducted. Results: MSI post-chromatic aberration correction found a significant interaction effect with age and retinal depth in IR, GR and BR (P < 0.05). The reflectance ratio of short/long wavelength shows a significant interaction effect (BR/IR, P < 0.01; average (BR,GR)/IR, P < 0.05) or trend GR/IR, P = 0.13 with advancing age and retinal depth. HSI also found a decrease in reflectance ratio with age (P < 0.0001), consistent with MSI. The effect size (Cohen's d) between HSI and MSI in the middle retina were comparable (P < 0.05). Conclusions: Confocal MSI can be achieved on a common commercial optical coherence tomography (OCT) device with off the shelf add-on features, and after chromatic aberration correction, enables depth-resolved spectral analysis. This offers advances over flood-illuminated HSI, which does not return depth information and requires custom made or dedicated equipment. Both MSI and HSI detect similar age-related retinal reflectivity changes. This development conceptually parallels the transition from two-dimensional fundus photography to three-dimensional OCT, offering potential enhancements for selected wavelengths.