Transient retinal phototropism (TRP) has been observed in rod photoreceptors activated by oblique visible light flashes. Time-lapse confocal microscopy and optical coherence tomography (OCT) revealed rod outer segment (ROS) movements as the physical source of TRP. However, the physiological source of TRP is still not well understood. In this study, concurrent TRP and electroretinogram (ERG) measurements disclosed a remarkably earlier onset time of the ROS movements (≤10 ms) than that (~38 ms) of the ERG a-wave. Furthermore, low sodium treatment reversibly blocked the photoreceptor ERG a-wave, which is known to reflect hyperpolarization of retinal photoreceptors, but preserved the TRP associated rod OS movements well. Our experimental results and theoretical analysis suggested that the physiological source of TRP might be attributed to early stages of phototransduction, before the hyperpolarization of retinal photoreceptors.
It is well established that major retinal diseases involve distortions of the retinal neural physiology and blood vascular
structures. However, the details of distortions in retinal neurovascular coupling associated with major eye diseases are
not well understood. In this study, a multi-modal optical coherence tomography (OCT) imaging system was developed
to enable concurrent imaging of retinal neural activity and vascular hemodynamics. Flicker light stimulation was applied
to mouse retinas to evoke retinal neural responses and hemodynamic changes. The OCT images were acquired
continuously during the pre-stimulation, light-stimulation, and post-stimulation phases. Stimulus-evoked intrinsic optical
signals (IOSs) and hemodynamic changes were observed over time in blood-free and blood regions, respectively. Rapid
IOSs change occurred almost immediately after stimulation. Both positive and negative signals were observed in
adjacent retinal areas. The hemodynamic changes showed time delays after stimulation. The signal magnitudes induced
by light stimulation were observed in blood regions and did not show significant changes in blood-free regions. These
differences may arise from different mechanisms in blood vessels and neural tissues in response to light stimulation.
These characteristics agreed well with our previous observations in mouse retinas. Further development of the multimodal
OCT may provide a new imaging method for studying how retinal structures and metabolic and neural functions
are affected by age-related macular degeneration (AMD), glaucoma, diabetic retinopathy (DR), and other diseases,
which promises novel noninvasive biomarkers for early disease detection and reliable treatment evaluations of eye
High resolution is important for sensitive detection of subtle distortions of retinal morphology at an early stage of eye diseases. We demonstrate virtually structured detection (VSD) as a feasible method to achieve in vivo super-resolution ophthalmoscopy. A line-scanning strategy was employed to achieve a super-resolution imaging speed up to 127 frames/s with a frame size of 512×512 pixels. The proof-of-concept experiment was performed on anesthetized frogs. VSD-based super-resolution images reveal individual photoreceptors and nerve fiber bundles unambiguously. Both image contrast and signal-to-noise ratio are significantly improved due to the VSD implementation.
Intrinsic optical signal (IOS) imaging promises a noninvasive method for advanced study and diagnosis of eye diseases. Before pursuing clinical applications, it is essential to understand anatomic and physiological sources of retinal IOSs and to establish the relationship between IOS distortions and eye diseases. The purpose of this study was designed to demonstrate the feasibility of in vivo IOS imaging of mouse models. A high spatiotemporal resolution spectral domain optical coherence tomography (SD-OCT) was employed for depth-resolved retinal imaging. A custom-designed animal holder equipped with ear bar and bite bar was used to minimize eye movements. Dynamic OCT imaging revealed rapid IOS from the photoreceptor’s outer segment immediately after the stimulation delivery, and slow IOS changes were observed from inner retinal layers. Comparative photoreceptor IOS and electroretinography recordings suggested that the fast photoreceptor IOS may be attributed to the early stage of phototransduction before the hyperpolarization of retinal photoreceptor.
Rod-dominated transient retinal phototropism (TRP) has been recently observed in freshly isolated mouse and frog retinas. Comparative confocal microscopy and optical coherence tomography revealed that the TRP was predominantly elicited from the rod outer segment (OS). However, the biophysical mechanism of rod OS dynamics is still unknown. Mouse and frog retinal slices, which displayed a cross-section of retinal photoreceptors and other functional layers, were used to test the effect of light stimulation on rod OSs. Time-lapse microscopy revealed stimulus-evoked conformational changes of rod OSs. In the center of the stimulated region, the length of the rod OS shrunk, while in the peripheral region, the rod OS swung toward the center region. Our experimental observation and theoretical analysis suggest that the TRP may reflect unbalanced rod disc-shape changes due to localized visible light stimulation.
Oblique light stimulation evoked transient retinal phototropism (TRP) has been recently detected in frog and mouse retinas. High resolution microscopy of freshly isolated retinas indicated that the TRP is predominated by rod photoreceptors. Comparative confocal microscopy and optical coherence tomography (OCT) revealed that the TRP predominantly occurred from the photoreceptor outer segment (OS). However, biophysical mechanism of rod OS change is still unknown. In this study, frog retinal slices, which open a cross section of retinal photoreceptor and other functional layers, were used to test the effect of light stimulation on rod OS. Near infrared light microscopy was employed to monitor photoreceptor changes in retinal slices stimulated by a rectangular-shaped visible light flash. Rapid rod OS length change was observed after the stimulation delivery. The magnitude and direction of the rod OS change varied with the position of the rods within the stimulated area. In the center of stimulated region the length of the rod OS shrunk, while in the peripheral region the rod OS tip swung towards center region in the plane perpendicular to the incident stimulus light. Our experimental result and theoretical analysis suggest that the observed TRP may reflect unbalanced disc-shape change due to localized pigment bleaching. Further investigation is required to understand biochemical mechanism of the observed rod OS kinetics. Better study of the TRP may provide a noninvasive biomarker to enable early detection of age-related macular degeneration (AMD) and other diseases that are known to produce retinal photoreceptor dysfunctions.
Intrinsic optical signal (IOS) imaging is a promising noninvasive method for advanced study and diagnosis of eye
diseases. Before pursuing clinical applications, more IOS studies employing animal models are necessary to establish the
relationship between IOS distortions and eye diseases. Ample mouse models are available for investigating the
relationship between IOS distortions and eye diseases. However, in vivo IOS imaging of mouse retinas is challenging
due to the small ocular lens (compared to frog eyes) and inevitable eye movements. We report here in vivo IOS imaging
of mouse retinas using a custom-designed functional OCT. The OCT system provided high resolution (3 μm) and high
speed (up to 500 frames/s) imaging of mouse retinas. An animal holder equipped with a custom designed ear bar and bite
bar was used to minimize eye movement due to breathing and heartbeats. Residual eye movement in OCT images was
further compensated by accurate image registration. Dynamic OCT imaging revealed rapid IOSs from photoreceptor
outer segments immediately (<10 ms) after the stimulation delivery, and unambiguous IOS changes were also observed
from inner retinal layers with delayed time courses compared to that of photoreceptor IOSs.
Intrinsic optical signal (IOS) imaging promises to be a noninvasive method for high-resolution examination of retinal physiology, which can advance the study and diagnosis of eye diseases. While specialized optical instruments are desirable for functional IOS imaging of retinal physiology, in depth understanding of multiple IOS sources in the complex retinal neural network is essential for optimizing instrument designs. We provide a brief overview of IOS studies and relationships in rod outer segment suspensions, isolated retinas, and intact eyes. Recent developments of line-scan confocal and functional optical coherence tomography (OCT) instruments have allowed in vivo IOS mapping of photoreceptor physiology. Further improvements of the line-scan confocal and functional OCT systems may provide a feasible solution to pursue functional IOS mapping of human photoreceptors. Some interesting IOSs have already been detected in inner retinal layers, but better development of the IOS instruments and software algorithms is required to achieve optimal physiological assessment of inner retinal neurons.