Rongwen Lu

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.

Dynamic near-infrared imaging reveals transient phototropic change in retinal rod photoreceptors

Abstract. Stiles–Crawford effect (SCE) is exclusively observed in cone photoreceptors, but why the SCE is absent in rod photoreceptors is still a mystery. In this study, we employed dynamic near infrared light imaging to monitor photoreceptor kinetics in freshly isolated frog and mouse retinas stimulated by oblique visible light flashes. It was observed that retinal rods could rapidly (onset: ∼10  ms for frog and 5 ms for mouse; time-to-peak: ∼200  ms for frog and 30 ms for mouse) shift toward the direction of the visible light, which might quickly compensate for the loss of luminous efficiency due to oblique illumination. In contrast, such directional movement was negligible in retinal cones. Moreover, transient rod phototropism could contribute to characteristic intrinsic optical signal (IOS). We anticipate that further study of the transient rod phototropism may not only provide insight into better understanding of the nature of vision but also promise an IOS biomarker for functional mapping of rod physiology at high resolution.

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.

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.

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.

A polarization-sensitive light field imager for multi-channel angular spectroscopy of light scattering in biological tissues.

BACKGROUND Angular spectroscopy of light scattering can be used for quantitative analysis of cellular and subcellular properties, and thus promises a noninvasive methodology for in vivo assessment cellular integrity to complement in vitro histological examination. Spatial information is essential for accurate identification of localized abnormalities. However, conventional angular spectroscopy systems only provide single-channel measurement, which suffers from poor spatial resolution or requires time-consuming scanning over extended area. The purpose of this study was to develop a multi-channel angular spectroscopy for light field imaging in biological tissues. MATERIALS AND METHODS A microlens array (MLA) (8×8) based light field imager for 64-channel angular spectroscopy was developed. A pair of crossed polarizers was employed for polarization-sensitive recording to enable quantitative measurement at high signal specificity and sensitivity. The polarization-sensitive light field imager enables rapid measurement of multiple sampling volumes simultaneously at 18 μm spatial-resolution and 3° angular-resolution. Comparative light field imaging and electrophysiological examination of freshly isolated and physiologically deteriorated lobster leg nerves have been conducted. RESULTS Two-dimensional (2D) polarization-sensitive scattering patterns of the fresh nerves were highly elliptical, while they gradually lost the ellipticity and became rotationally symmetric (i.e., circular) as the nerves physiologically deteriorated due to repeated electrical stimulations. Characterized parameters, i.e., the ellipticity and the scattering intensity, rendered spatially various characteristics such as different values and deteriorating rates. CONCLUSIONS The polarization-sensitive light field imager is able to provide multi-channel angular spectroscopy of light scattering with both spatial and angular resolutions. The light scattering properties of nerves are highly dependent on the orientation of nerves and their physiological status. Further development of polarization-sensitive multi-channel angular spectroscopy may promise a methodology for rapid and reliable identification of localized abnormalities in biological tissues.