Super-resolution ophthalmoscopy: Virtually structured detection for resolution improvement in retinal imaging.

Quantitative retinal imaging is essential for advanced study and clinical management of eye diseases. However, spatial resolution of retinal imaging has been limited due to available numerical aperture and optical aberration of the ocular optics. Structured illumination microscopy has been established to break the diffraction-limit resolution in conventional light microscopy. However, practical implementation of structured illumination microscopy for in vivo ophthalmoscopy of the retina is challenging due to inevitable eye movements that can produce phase artifacts. Recently, we have demonstrated the feasibility of using virtually structured detection as one alternative to structured illumination microscopy for super-resolution imaging. By providing the flexibility of digital compensation of eye movements, the virtually structured detection provides a feasible, phase-artifact-free strategy to achieve super-resolution ophthalmoscopy. In this article, we summarize the technical rationale of virtually structured detection, and its implementations for super-resolution imaging of freshly isolated retinas, intact animals, and awake human subjects.

Functional optical coherence tomography enables in vivo optoretinography of photoreceptor dysfunction due to retinal degeneration.

Stimulus-evoked intrinsic optical signal (IOS), which occurs almost immediately after the onset of retinal stimulus has been observed in retinal photoreceptors, promises to be a unique biomarker for objective optoretinography (ORG) of photoreceptor function. We report here the first-time in vivo ORG detection of photoreceptor dysfunction due to retinal degeneration. A custom-designed optical coherence tomography (OCT) was employed for longitudinal ORG monitoring of photoreceptor-IOS distortions in retinal degeneration mice. Depth-resolved OCT analysis confirmed the outer segment (OS) as the physical source of the photoreceptor-IOS. Comparative ERG measurement verified the phototransduction activation as the physiological correlator of the photoreceptor-IOS. Histological examination revealed disorganized OS discs, i.e. the pathological origin of the photoreceptor-IOS distortion.

Contact-free trans-pars-planar illumination enables snapshot fundus camera for nonmydriatic wide field photography.

In conventional fundus photography, trans-pupillary illumination delivers illuminating light to the interior of the eye through the peripheral area of the pupil, and only the central part of the pupil can be used for collecting imaging light. Therefore, the field of view of conventional fundus cameras is limited, and pupil dilation is required for evaluating the retinal periphery which is frequently affected by diabetic retinopathy (DR), retinopathy of prematurity (ROP), and other chorioretinal conditions. We report here a nonmydriatic wide field fundus camera employing trans-pars-planar illumination which delivers illuminating light through the pars plana, an area outside of the pupil. Trans-pars-planar illumination frees the entire pupil for imaging purpose only, and thus wide field fundus photography can be readily achieved with less pupil dilation. For proof-of-concept testing, using all off-the-shelf components a prototype instrument that can achieve 90° fundus view coverage in single-shot fundus images, without the need of pharmacologic pupil dilation was demonstrated.

Functional optical coherence tomography of neurovascular coupling interactions in the retina.

Quantitative evaluation of retinal neurovascular coupling is essential for a better understanding of visual function and early detection of eye diseases. However, there is no established method to monitor coherent interactions between stimulus-evoked neural activity and hemodynamic responses at high resolution. Here, we report a multimodal functional optical coherence tomography (OCT) imaging methodology to enable concurrent intrinsic optical signal (IOS) imaging of stimulus-evoked neural activity and hemodynamic responses at capillary resolution. OCT angiography guided IOS analysis was used to separate neural-IOS and hemodynamic-IOS changes in the same retinal image sequence. Frequency flicker stimuli evoked neural-IOS changes in the outer retina; that is, photoreceptor layer, first and then in the inner retina, including outer plexus layer (OPL), inner plexiform layer (IPL), and ganglion cell layer (GCL), which were followed by hemodynamic-IOS changes primarily in the inner retina; that is, OPL, IPL, and GCL. Different time courses and signal magnitudes of hemodynamic-IOS responses were observed in blood vessels with various diameters.

Concurrent OCT imaging of stimulus evoked retinal neural activation and hemodynamic responses.

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 multi-modal 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 diseases.

Enhancement of intrinsic optical signal recording with split spectrum optical coherence tomography.

Functional optical coherence tomography (OCT) of stimulus-evoked intrinsic optical signal (IOS) promises to be a new methodology for high-resolution mapping of retinal neural dysfunctions. However, its practical applications for non-invasive examination of retinal function have been hindered by the low signal-to-noise ratio (SNR) and small magnitude of IOSs. Split spectrum amplitude-decorrelation has been demonstrated to improve the image quality of OCT angiography. In this study, we exploited split spectrum strategy to improve the sensitivity of IOS recording. The full OCT spectrum was split into multiple spectral bands and IOSs from each sub-band were calculated separately and then combined to generate a single IOS image sequence. The algorithm was tested on in vivo images of frog retinas. It significantly improved both IOS magnitude and SNR, which are essential for practical applications of functional IOS imaging.

Comparative investigation of stimulus-evoked rod outer segment movement and retinal electrophysiological activity.

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.

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.

In vivo super-resolution retinal imaging through virtually structured detection.

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.

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

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

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 in rod photoreceptors.

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.

Biophysical mechanism of transient retinal phototropism in rod photoreceptors.

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.

In vivo intrinsic optical signal imaging of mouse retinas.

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 imaging of retinal physiology: a review.

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.

Promotion of Metastasis-associated Gene Expression in Survived PANC-1 Cells Following Trichostatin A Treatment.

OBJECTIVES: Histone deacetylase inhibitors represent a promising class of potential anticancer agents for the treatment of human malignancies. In this study, the effects of trichostatin A (TSA) on apoptosis, metastasis-associated gene expression, and activation of the Notch pathway in human pancreatic cancer cell lines were investigated. METHODS: After treatment with TSA, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide] assay, Hoechst 33258 staining, and flow cytometry. Moreover, RT-PCR and western blot analyses were performed to measure the expression levels of apoptosis-associated genes (Bcl-2, Bax, and caspase-3), metastasis-associated genes (E-cadherin, vimentin, and matrix metalloproteinases), and Notch pathway activation (Notch intracellular domain, NICD). The levels of matrix metalloproteinase 2 and NICD were also semi-quantified by immunoassay. RESULTS: Following treatment with TSA for 24 h, PANC-1, SW1990, and MIATACA-2 cells exhibited cell death. The MTT assay revealed that TSA significantly decreased cell viability in a dose-dependent manner in PANC-1 cells. The Hoechst 33258 staining and flow cytometry results evidenced a significant increase in PANC-1 cell apoptosis following TSA treatment. The expression levels of Bax and caspase-3 were increased significantly, whereas Bcl-2 was down-regulated after TSA treatment. In the PANC-1 cells that survived after TSA treatment, the expression levels of vimentin, E-cadherin, and MMP genes were altered by the promotion of potential metastasis and increased expression of NICD. CONCLUSIONS: TSA can induce apoptosis of pancreatic cancer cells. In addition, the up-regulation of metastasis-related genes and the activation of the Notch pathway in the survived PANC-1 cells may be associated with a too-low level of TSA or resistance to TSA.

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.

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.

Super-Resolution Scanning Laser Microscopy Based on Virtually Structured Detection.

Light microscopy plays a key role in biological studies and medical diagnosis. The spatial resolution of conventional optical microscopes is limited to approximately half the wavelength of the illumination light as a result of the diffraction limit. Several approaches-including confocal microscopy, stimulated emission depletion microscopy, stochastic optical reconstruction microscopy, photoactivated localization microscopy, and structured illumination microscopy-have been established to achieve super-resolution imaging. However, none of these methods is suitable for the super-resolution ophthalmoscopy of retinal structures because of laser safety issues and inevitable eye movements. We recently experimentally validated virtually structured detection (VSD) as an alternative strategy to extend the diffraction limit. Without the complexity of structured illumination, VSD provides an easy, low-cost, and phase artifact-free strategy to achieve super-resolution in scanning laser microscopy. In this article we summarize the basic principles of the VSD method, review our demonstrated single-point and line-scan super-resolution systems, and discuss both technical challenges and the potential of VSD-based instrumentation for super-resolution ophthalmoscopy of the retina.

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.