Purpose To assess the ability of optical coherence tomography angiography (OCTA) to image the retinal middle capillary plexus (MCP), and to characterize the MCP as a unique vascular network separate from the superficial (SCP) and deep capillary plexus (DCP). Methods Healthy and diabetic eyes were imaged using the Avanti XR OCTA instrument (Optovue Inc, Fremont, California, USA). Using manual segmentation of the retinal layers, we generated en face angiograms to distinguish the three capillary plexuses (SCP, MCP, DCP). Results In healthy eyes, arterioles gave rise to distinct branches in the MCP, and venules gave rise to prominent vortex like branches in the DCP. The foveal avascular zone (FAZ) was most well-defined at the level of the MCP, and had a larger area in the DCP. In diabetic eyes, the three capillary plexuses showed varying degrees of non-perfusion, including variable shapes and extent of the FAZ, with loss of border integrity at the MCP. Microaneurysms appeared in all of the three capillary plexuses. Conclusions Using customized segmentation analysis in OCTA, we demonstrate that the MCP is qualitatively and functionally distinct from the SCP and DCP, which may help clarify the pathogenesis of different middle retinal ischemic entities and provide new insights into retinal ischemia in diabetic retinopathy.

Purpose To study choriocapillaris blood flow in age-related macular degeneration (AMD) using optical coherence tomography angiography (OCTA) and study its correlation to vision (VA) in eyes with reticular pseudodrusen (RPD) versus those with drusen without RPD (drusen). Design Cross-sectional study Methods Patients with either drusen or RPD in early AMD underwent OCTA imaging of the superior, inferior, and/or nasal macula. We quantified “percent choriocapillaris area of non-perfusion” (PCAN) in eyes with RPD versus those with drusen. We assessed the repeatability of PCAN and its correlations with VA. Results Twenty-nine eyes of 26 patients with RPD and 21 eyes of 16 age-matched AMD patients with drusen were included. Qualitatively, the choriocapillaris in areas with RPD showed focal dark regions without flow signal on OCTA (non-perfusion). The repeatability coefficient of PCAN was 0.49%. Eyes with RPD had significantly greater PCAN compared to eyes with drusen (7.31% and 3.88%, respectively; P < 0.001). We found a significant correlation between PCAN and VA for the entire dataset (r = 0.394, P = 0.005). When considering eyes with RPD separately, this correlation was stronger (r = 0.474, P = 0.009) but lost significance when considering eyes with drusen separately (r = 0.175, P = 0.45). Conclusions Eyes with RPD have significantly larger areas of choriocapillaris non-perfusion compared to eyes with drusen and no RPD. The correlation between PCAN and VA in this RPD population provides a potential mechanistic explanation for vision compromise in RPD compared to other forms of drusen in AMD.

Capitalizing on the optical absorption of hemoglobin, photoacoustic microscopy (PAM) is uniquely capable of anatomical and functional characterization of the intact microcirculation in vivo. However, PAM of the metabolic rate of oxygen (MRO₂) at the microscopic level remains an unmet challenge, mainly due to the inability to simultaneously quantify microvascular diameter, oxygen saturation of hemoglobin (sO₂), and blood flow at the same spatial scale. To fill this technical gap, we have developed a multi-parametric PAM platform. By analyzing both the sO₂-encoded spectral dependence and the flow-induced temporal decorrelation of photoacoustic signals generated by the raster-scanned mouse ear vasculature, we demonstrated-for the first time-simultaneous wide-field PAM of all three parameters down to the capillary level in vivo.

PurposeTo identify the microvascular changes associated with paracentral acute middle maculopathy (PAMM) and acute macular neuroretinopathy (AMN) and to improve our understanding of the relevant involvement of the three retinal capillary plexuses using projection-resolved optical coherence tomography angiography (PR-OCTA).MethodsThis was a retrospective study of 18 eyes with AMN or PAMM imaged with OCTA. We used cross-sectional PR-OCTA to localize reduced flow signal to the superficial (SCP), middle (MCP), or deep capillary plexus (DCP) or choriocapillaris that corresponded to inner retinal PAMM or outer retinal AMN lesions on OCT.ResultsFive eyes with AMN showed outer retinal disruption on OCT associated with reduced DCP flow signal. All three eyes with AMN and follow-up had recovery of DCP flow. Thirteen eyes with PAMM showed middle retinal disruption on OCT associated with reduced flow signal in both the MCP and DCP. Of these, five also had reduced flow signal in the SCP. All 10 eyes with PAMM and follow-up showed variable recovery of flow signal in one or more plexuses. PAMM reperfusion was primarily arterial in nature. Three eyes with PAMM and no evidence of MCP reperfusion experienced severe thinning of the inner nuclear layer (INL), while seven eyes with robust MCP flow signal recovery showed relative preservation of INL thickness.ConclusionsUsing PR-OCTA, we found that AMN was associated with reduced DCP flow signal, while PAMM was associated with reduced MCP and DCP flow signal and occasionally the SCP. The MCP appears to be important in sustaining INL thickness in these eyes.

The mouse laser-induced choroidal neovascularization (CNV) model has been a crucial mainstay model for neovascular age-related macular degeneration (AMD) research. By administering targeted laser injury to the RPE and Bruch's membrane, the procedure induces angiogenesis, modeling the hallmark pathology observed in neovascular AMD. First developed in non-human primates, the laser-induced CNV model has come to be implemented into many other species, the most recent of which being the mouse. Mouse experiments are advantageously more cost-effective, experiments can be executed on a much faster timeline, and they allow the use of various transgenic models. The miniature size of the mouse eye, however, poses a particular challenge when performing the procedure. Manipulation of the eye to visualize the retina requires practice of fine dexterity skills as well as simultaneous hand-eye-foot coordination to operate the laser. However, once mastered, the model can be applied to study many aspects of neovascular AMD such as molecular mechanisms, the effect of genetic manipulations, and drug treatment effects. The laser-induced CNV model, though useful, is not a perfect model of the disease. The wild-type mouse eye is otherwise healthy, and the chorio-retinal environment does not mimic the pathologic changes in human AMD. Furthermore, injury-induced angiogenesis does not reflect the same pathways as angiogenesis occurring in an age-related and chronic disease state as in AMD. Despite its shortcomings, the laser-induced CNV model is one of the best methods currently available to study the debilitating pathology of neovascular AMD. Its implementation has led to a deeper understanding of the pathogenesis of AMD, as well as contributing to the development of many of the AMD therapies currently available.

In recent years, advances in optical coherence tomography (OCT) techniques have increased our understanding of diabetic retinopathy, an important microvascular complication of diabetes. OCT angiography is a non-invasive method that visualizes the retinal vasculature by detecting motion contrast from flowing blood. Visible-light OCT shows promise as a novel technique for quantifying retinal hypoxia by measuring the retinal oxygen delivery and metabolic rates. In this article, we discuss recent insights provided by these techniques into the vascular pathophysiology of diabetic retinopathy. The next milestones for these modalities are large multicenter studies to establish consensus on the most reliable and consistent outcome parameters to study diabetic retinopathy.

We have developed fully motorized optical-resolution photoacoustic microscopy (OR-PAM), which integrates five complementary scanning modes and simultaneously provides a high imaging speed and a wide field of view (FOV) with 2.6 µm lateral resolution. With one-dimensional (1D) motion-mode mechanical scanning, we measured the blood flow through a cross section of a blood vessel in vivo. With two-dimensional (2D) optical scanning at a laser repetition rate of 40 kHz, we achieved a 2 kHz B-scan rate over a range of 50 µm with 20 A-lines, and 50 Hz volumetric-scan rate over a FOV of 50×50 µm2 with 400 A-lines, which enabled real-time tracking of cellular dynamics in vivo. With synchronized 1D optical and 2D mechanical hybrid scanning, we imaged a 10×8 mm2 FOV within three minutes, which is 20 times faster than the conventional mechanical scan in our second-generation OR-PAM. With three-dimensional mechanical contour scanning, we maintained the optimal signal-to-noise ratio and spatial resolution of OR-PAM while imaging objects with uneven surfaces, which is essential for quantitative studies.

Semiconductor nanocrystals and their variants are widely used in biological research as fluorescent probes. Their unique characteristics, such as intense brightness, tunable emission properties, and resistance to photobleaching, make them ideal candidates for single-molecule imaging and tracking with localization precision far beyond the diffraction limit. Their fluorescence polarization states and emission spectra can be further utilized to probe changes in their mechanical properties and residing nanoenvironments. We developed a three-dimensional (3D), polarization-sensitive, spectroscopic photon localization microscopy (3D-Polar-SPLM) that enables parallel 3D tracking of individual quantum rods (QRs) while simultaneously capturing their fluorescence spectra and polarization states. Using 3D-Polar-SPLM, we spatially localized individual QRs with a lateral localization precision of 8 nm and an axial localization precision of 35 nm. In addition, we achieved a spectral resolution of 2 nm and a polarization angle measuring precision of 8 degrees. The spectral profile of the fluorescence emission provided a particle-specific signature for identifying individual QRs among the heterogeneous population, which significantly improved the fidelity in parallel 3D tracking of multiple QRs. We envision that this technology will provide new possibilities to reveal the real-time molecular dynamics of biological processes.