Brian T. Soetikno

Simultaneous photoacoustic microscopy of microvascular anatomy, oxygen saturation, and blood flow.

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.

CHARACTERIZATION of the MIDDLE CAPILLARY PLEXUS USING OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY in HEALTHY and DIABETIC EYES

Purpose: To assess the ability of optical coherence tomography angiography to image the retinal middle capillary plexus (MCP), and to characterize the MCP as a unique vascular network separate from the superficial and deep capillary plexus (DCP). Methods: Healthy and diabetic eyes were imaged using the Avanti XR optical coherence tomography angiography instrument (Optovue Inc, Fremont, CA). Using manual segmentation of the retinal layers, the authors generated en face angiograms to distinguish the three capillary plexuses (superficial capillary plexus, 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 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 nonperfusion, including variable shapes and extent of the foveal avascular zone, with loss of border integrity at the MCP. Microaneurysms appeared in all the three capillary plexuses. Conclusion: Using customized segmentation analysis in optical coherence tomography angiography, the authors demonstrate that the MCP is qualitatively and functionally distinct from the superficial capillary plexus and DCP, which may help clarify the pathogenesis of different middle retinal ischemic entities and provide new insights into retinal ischemia in diabetic retinopathy.

Fully motorized optical-resolution photoacoustic microscopy

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  μm × 50  μm 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  mm × 8  mm 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.

Inner retinal oxygen metabolism in the 50/10 oxygen-induced retinopathy model

Retinopathy of prematurity (ROP) represents a major cause of childhood vision loss worldwide. The 50/10 oxygen-induced retinopathy (OIR) model mimics the findings of ROP, including peripheral vascular attenuation and neovascularization. The oxygen metabolism of the inner retina has not been previously explored in this model. Using visible-light optical coherence tomography (vis-OCT), we measured the oxygen saturation of hemoglobin and blood flow within inner retinal vessels, enabling us to compute the inner retinal oxygen delivery (irDO2) and metabolic rate of oxygen (irMRO2). We compared these measurements between age-matched room-air controls and rats with 50/10 OIR on postnatal day 18. To account for a 61% decrease in the irDO2 in the OIR group, we found an overall statistically significant decrease in retinal vascular density affecting the superficial and deep retinal vascular capillary networks in rats with OIR compared to controls. Furthermore, matching the reduced irDO2, we found a 59% decrease in irMRO2, which we correlated with a statistically significant reduction in retinal thickness in the OIR group, suggesting that the decreased irMRO2 was due to decreased neuronal oxygen utilization. By exploring these biological and metabolic changes in great detail, our study provides an improved understanding of the pathophysiology of OIR model.

Imaging hemodynamic response after ischemic stroke in mouse cortex using visible-light optical coherence tomography

Visible-light optical coherence tomography (Vis-OCT) is an emerging technology that can image hemodynamic response in microvasculature. Vis-OCT can retrieve blood oxygen saturation (sO2) mapping using intrinsic optical absorption contrast while providing high-resolution anatomical vascular structures at the same time. To improve the accuracy of Vis-OCT oximetry on vessels embedded in highly scattering medium, i.e., brain cortex, we developed and formulated a novel dual-depth sampling and normalization strategy that allowed us to minimize the detrimental effect of ubiquitous tissue scattering. We applied our newly developed approach to monitor the hemodynamic response in mouse cortex after focal photothrombosis. We observed vessel dilatation, which was negatively correlated with the original vessel diameter, in the penumbra region. The sO2 of vessels in the penumbra region also dropped below normal range after focal ischemia.

Visible-Light Optical Coherence Tomography Angiography for Monitoring Laser-Induced Choroidal Neovascularization in Mice.

Purpose This study sought to determine the earliest time-point at which evidence of choroidal neovascularization (CNV) could be detected with visible-light optical coherence tomography angiography (vis-OCTA) in a mouse model of laser-induced CNV. Methods Visible light-OCTA was used to study laser-induced CNV at different time-points after laser injury to monitor CNV development and measure CNV lesion size. Measurements obtained from vis-OCTA angiograms were compared with histopathologic measurements from isolectin-stained choroidal flatmounts. Results Choroidal neovascularization area measurements between the vis-OCTA system and isolectin-stained choroidal flatmounts were significantly different in area for days 2 to 4 postlaser injury, and were not significantly different in area for days 5, 7, and 14. Choroidal neovascularization area measurements taken from the stained flatmounts were larger than their vis-OCTA counterparts for all time-points. Both modalities showed a similar trend of CNV size increasing from the day of laser injury until a peak of day 7 postlaser injury and subsequently decreasing by day 14. Conclusions The earliest vis-OCTA can detect the presence of aberrant vessels in a mouse laser-induced CNV model is 5 days after laser injury. Visible light-OCTA was able to visualize the maximum of the CNV network 7 days postlaser injury, in accordance with choroidal flatmount immunostaining. Visible light-OCTA is a reliable tool in both detecting the presence of CNV development, as well as accurately determining the size of the lesion in a mouse laser-induced CNV model.

Optical-resolution photoacoustic microscopy of ischemic stroke

A major obstacle in understanding the mechanism of ischemic stroke is the lack of a tool to noninvasively or minimally invasively monitor cerebral hemodynamics longitudinally. Here, we applied optical-resolution photoacoustic microscopy (OR-PAM) to longitudinally study ischemic stroke induced brain injury in a mouse model with transient middle cerebral artery occlusion (MCAO). OR-PAM showed that, during MCAO, the average hemoglobin oxygen saturation (sO2) values of feeder arteries and draining veins within the stroke core region dropped ~10% and ~34%, respectively. After reperfusion, arterial sO2 recovered back to the baseline; however, the venous sO2 increased above the baseline value by ~7%. Thereafter, venous sO2 values were close to the arterial sO2 values, suggesting eventual brain tissue infarction.

A Mouse Model for Laser-induced Choroidal Neovascularization

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 Bruchs 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.

Vessel Segmentation Analysis of Ischemic Stroke Images Acquired with Photoacoustic Microscopy

We have applied optical-resolution photoacoustic microscopy (OR-PAM) for longitudinal monitoring of cerebral metabolism through the intact skull of mice before, during, and up to 72 hours after a 1-hour transient middle cerebral artery occlusion (tMCAO). The high spatial resolution of OR-PAM enabled us to develop vessel segmentation techniques for segment-wise analysis of cerebrovascular responses.

OCT angiography and visible-light OCT in diabetic retinopathy

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.