Benquan Wang

Functional Optical Coherence Tomography Enables In Vivo Physiological Assessment of Retinal Rod and Cone Photoreceptors

Transient intrinsic optical signal (IOS) changes have been observed in retinal photoreceptors, suggesting a unique biomarker for eye disease detection. However, clinical deployment of IOS imaging is challenging due to unclear IOS sources and limited signal-to-noise ratios (SNRs). Here, by developing high spatiotemporal resolution optical coherence tomography (OCT) and applying an adaptive algorithm for IOS processing, we were able to record robust IOSs from single-pass measurements. Transient IOSs, which might reflect an early stage of light phototransduction, are consistently observed in the photoreceptor outer segment almost immediately (<4 ms) after retinal stimulation. Comparative studies of dark- and light-adapted retinas have demonstrated the feasibility of functional OCT mapping of rod and cone photoreceptors, promising a new method for early disease detection and improved treatment of diseases such as age-related macular degeneration (AMD) and other eye diseases that can cause photoreceptor damage.

Functional optical coherence tomography reveals transient phototropic change of photoreceptor outer segments.

Dynamic near infrared microscopy has revealed transient retinal phototropism (TRP) correlated with oblique light stimulation. Here, by developing a hybrid confocal microscopy and optical coherence tomography (OCT), we tested sub-cellular source of the TRP in living frog retina. Dynamic confocal microscopy and OCT consistently revealed photoreceptor outer segments as the anatomic source of the TRP. Further investigation of the TRP can provide insights in better understanding of Stiles-Crawford effect (SCE) on rod and cone systems, and may also promise an intrinsic biomarker for early detection of eye diseases that can produce photoreceptor dysfunction.

Intrinsic optical signal imaging of retinal physiology: a review

Abstract. 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.

En face optical coherence tomography of transient light response at photoreceptor outer segments in living frog eyecup

This study was designed to test the feasibility of en face mapping of the transient intrinsic optical signal (IOS) response at photoreceptor outer segments and to assess the effect of spatial resolution on functional IOS imaging of retinal photoreceptors. A line-scan optical coherence tomography (LS-OCT) was constructed to achieve depth-resolved functional IOS imaging of living frog eyecups. Rapid en face OCT revealed transient IOS almost immediately (<3 ms) after the onset of visible light flashes at photoreceptor outer segments. Quantitative analysis indicated that the IOS kinetics may reflect dynamics of G-protein binding and releasing in early phases of visual transduction, and high resolution is essential to differentiate positive and negative IOS changes in adjacent locations.

Stimulus-evoked outer segment changes in rod photoreceptors

Abstract. 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.

In vivo optical coherence tomography of stimulus-evoked intrinsic optical signals in mouse retinas

Abstract. 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.

Rapid super-resolution line-scanning microscopy through virtually structured detection.

Virtually structured detection (VSD) has been demonstrated to break the diffraction limit in scanning laser microscopy (SLM). VSD provides an easy, low-cost, and phase-artifact-free strategy to achieve super-resolution imaging. However, practical application of this method is challenging due to a limited image acquisition speed. We report here the combination of VSD and line-scanning microscopy (LSM) to improve the image acquisition speed. A motorized dove prism was used to achieve automatic control of four-angle (i.e., 0°, 45°, 90°, and 135°) scanning, thus ensuring isotropic resolution improvement. Both an optical resolution target and a living frog eyecup were used to verify resolution enhancement.

Optical coherence tomography angiography of stimulus evoked hemodynamic responses in individual retinal layers

Blood flow changes are highly related to neural activities in the retina. It has been reported that neural activity increases when flickering light stimulation of the retina is used. It is known that blood flow changes with flickering light stimulation can be altered in patients with vascular disease and that measurement of flicker-induced vasodilatation is an easily applied tool for monitoring functional microvascular alterations. However, details of distortions in retinal neurovascular coupling associated with major eye diseases are not well understood due to the limitation of existing techniques. In this study, flickering light stimulation was applied to mouse retinas to investigate stimulus evoked hemodynamic responses in individual retinal layers. A spectral domain optical coherence tomography (OCT) angiography imaging system was developed to provide dynamic mapping of hemodynamic responses in the ganglion cell layer, inner plexiform layer, outer plexiform layer and choroid layer before, during and after flickering light stimulation. Experimental results showed hemodynamic responses with different magnitudes and time courses in individual retinal layers. We anticipate that the dynamic OCT angiography of stimulus evoked hemodynamic responses can greatly foster the study of neurovascular coupling mechanisms in the retina, promising new biomarkers for retinal disease detection and diagnosis.

Stimulus-evoked outer segment changes occur before the hyperpolarization of retinal photoreceptors

Transient retinal phototropism (TRP) has been predominantly observed in rod photoreceptors activated by oblique visible light stimulation. Dynamic confocal microscopy and optical coherence tomography (OCT) have revealed rod outer segment (ROS) movement as the physical source of TRP. However, the physiological source of ROS movement is still not well understood. In this study, concurrent near-infrared imaging of TRP and electroretinogram (ERG) measurement of retinal electrophysiology revealed that ROS movement occurs before the onset of the ERG a-wave, which is known to reflect the hyperpolarization of retinal photoreceptors. Moreover, substitution of normal superfusing medium with low-sodium medium reversibly blocked the photoreceptor ERG a-wave, but largely preserved the stimulus-evoked ROS movements. Our experimental results and theoretical analysis indicate that early, disc-based stages of the phototransduction cascade, which occur before the hyperpolarization of retinal photoreceptors, contribute to the TRP associated ROS movement.

Breaking diffraction limit of lateral resolution in optical coherence tomography.

Quantitative imaging of biomedical specimens is essential in biomedical study and diagnosis. Given excellent capability in three-dimensional (3D) imaging, optical coherence tomography (OCT) has been extensively used in ophthalmic imaging, vascular medicine, dermatological study, etc. Lateral resolution of the OCT is light diffraction limited, which precludes the feasibility of quantitative assessment of individual cells. In this paper, we demonstrated the feasibility of breaking diffraction-limit in OCT imaging through virtually structured detection (VSD). OCT examination of optical resolution target verified resolution doubling in the VSD based OCT imaging. Super-resolution OCT identification of individual frog photoreceptors was demonstrated to verify the potential of resolution enhancement in retinal imaging. We anticipate that further development of the VSD based OCT promises an easy, low cost strategy to achieve sub-cellular resolution tomography of the retina and other biological systems.