Quantitative Investigation of Stimulus-evoked Intrinsic Optical Signal Change in Retinal Photoreceptors

Transient intrinsic optical signal (IOS) responses have been observed in stimulus activated retinal photoreceptors. Functional IOS imaging of retinal photoreceptors promises a high spatio-temporal resolution method for objective assessment of retinal physiology, allowing early diagnoses of retinal diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP) that are known to produce photoreceptor dysfunctions. However, the physiological and anatomic mechanisms of the photoreceptor-IOS are still not well understood. Transient morphological perturbations, i.e., phototropism and shrinkage, of photoreceptor outer segment (OS) were observed when an oblique visible stimulus was applied. The spatio-temporal dynamics of these transient structural changes of OS were highly correlated with that of the stimulus-evoked photoreceptor-IOS responses. Therefore, the morphological perturbation of OS is speculated as an important contributor to the photoreceptor-IOS. This dissertation is designed to investigate the physical and physiological source of the light-induced photoreceptor outer segment changes and their spatiotemporal characteristics. The phototransduction-related physiological pathways of the light-induced the OS changes was first investigated with time-lapse light microscopy. Comparative study of physiological-solution-perfused retinas disclosed that the structural changes of OS had an origin earlier than the hyperpolarization of the OS membrane in the phototransduction cascade. Further studies involved mouse model with retinal degeneration indicated that the rapid IOS had a physiological origin in the early, disc-based stage of the phototransduction cascade. Transmission electron microscopy of dark- and light-adapted retinas was used to demonstrate the light-induced structure change of photoreceptor OSs at a subcellular level. The results demonstrated that the reduction of the inter-disc space may be correlated with the physical shrinkage of the light stimulated OSs. To verify the stimulus-evoked OS changes in vivo, a virtually-structural-detection (VSD) based super-resolution ophthalmoscopy and a line-scan modality was used to provide unambiguous identification of photoreceptors with a milli-second level temporal resolution. A robust transient retinal phototropism was observed in the living frog eyes and its temporal dynamics showed a tight correlation with the phototransduction. To further investigate the IOS responses in the human eye, an imaging technology with high spatial and temporal resolution is necessary considering the aberration and the involuntary movement of the human eye. A VSD-based super-resolution ophthalmoscope with a line-scan imaging modality was designed and constructed for in vivo human retinal imaging. Unambiguous identification of photoreceptor mosaic was achieved with cellular-level spatial resolution and millisecond-level temporal resolution. The successful demonstration of human retinal photoreceptors paves the road for the in vivo investigation of human photoreceptor-IOS in the future.