Super-resolution ophthalmoscopy for in vivo retinal imaging

描述(申请人提供)：这个为期3年的R01项目是为了探索一种新的超分辨率扫描激光眼底镜(SLO)方法。我们已经通过虚拟结构检测(VSD)展示了扫描激光显微镜(SLM)和光学相干层析成像(OCT)的分辨率翻倍。我们建议将VSD扩展到用于活体视网膜成像的超分辨率眼底镜中。在没有结构照明显微镜(SIM)的复杂性的情况下，VSD提供了一种简单、低成本和无相位伪影的策略来实现超分辨率成像。然而，VSD在活体视网膜成像中的可部署应用受到我们原型仪器有限的帧速度(每帧40秒)的挑战。由于眼球运动导致的帧内模糊，如此低的速度对于活体视网膜成像来说是无法忍受的。这里我们提出了结合快速行扫描策略和精确的图像配准来补偿眼睛运动。本项目的第一个目标是构建一台行扫描超分辨率SLO。这一目标的成功标准是生产出一种能够以10ms的帧速度进行超分辨率成像的仪器。为了实现超分辨率重建所需的三个角度(0度、30度和60度)扫描的快速变化，将组装一个振镜。对青蛙眼睛的初步行扫描成像表明，在10ms的帧速下，帧内模糊可以忽略不计；而逐帧运动可以通过精确的图像配准来校正。第二个目标是对所建议的仪器进行体外和体内验证。在活体实验之前，数值孔径NA=0.25的10倍物镜将用于20纳米纳米颗粒的超分辨率显微镜。体外实验是为了验证超分辨率仪器的空间分辨率。该项目的第二个目标是验证用于麻醉动物(青蛙)体内视网膜成像的超分辨率SLO。对麻醉青蛙进行活体成像，以量化超分辨率检眼镜和常规SLO的分辨率差异。第三个目的是验证完整动物的瞬时杆状向光性(Trp)。我们最近在新分离的小鼠和青蛙视网膜上展示了Trp。Trp的体内验证将提供一种光学生物标记物，适用于细胞分辨率的杆状生理功能标记物。该项目的成功终点是证明对完整动物进行超分辨率眼底镜检查的可行性。在该项目的下一阶段，超分辨眼底镜将用于研究正常动物中Trp的生物物理机制，并表征感光细胞变性转基因动物中Trp的异常修饰。此外，我们还计划将自适应光学与所建议的仪器相结合，在该项目的后续阶段对人体对象进行超分辨率眼底镜检查。

DESCRIPTION (provided by applicant): This 3-year R01 project is to explore a new methodology for super-resolution scanning laser ophthalmoscopy (SLO). We have demonstrated resolution-doubling in scanning laser microscopy (SLM) and optical coherence tomography (OCT) through virtually structured detection (VSD). We propose here to extend the VSD into super-resolution ophthalmoscopy for in vivo retinal imaging. Without the complexity of structured illumination microscopy (SIM), the VSD provides an easy, low-cost and phase-artifact free strategy to achieve super- resolution imaging. However, deployable application of the VSD for in vivo retinal imaging is challenged by limited frame-speed (40s per frame) of our prototype instrument. Such low speed is intolerable for in vivo retinal imaging because of within-frame blur due to eye movements. We propose here to combine rapid line-scan strategy and accurate image registration to compensate for eye movements. The first aim of this project is to construct a line-scan super-resolution SLO. Success criterion of this aim is to produce an instrument that enables super-resolution imaging at 10 ms frame-speed. In order to achieve rapid change of three-angle (0o, 30o and 60o) scanning required for super-resolution reconstruction, a galvo-dove-prism will be assembled. Preliminary line-scan imaging of frog eyes indicates that within-frame blur can be ignored at 10 ms frame- speed; while frame-by-frame movement can be corrected by accurate image registration. The second aim is to conduct both ex vivo and in vivo validation of the proposed instrument. Before in vivo experiments, a 10x objective with numeric aperture NA=0.25 will be used for super-resolution microscopy of 20 nm nanoparticles. The ex vivo experiment is designed to verify spatial resolution of the super-resolution instrument. The second aim of this project is to validate the super-resolution SLO for in vivo retinal imaging of anesthetized animals (frogs). In vivo imaging of anesthetized frog will be conducted to quantify resolution difference in super- resolution ophthalmoscopy and conventional SLO. The third aim to verify transient rod phototropism (TRP) in intact animals. We have recently demonstrated TRP in freshly isolated mouse and frog retinas. In vivo verification of the TRP will provide an optical biomarker suitable for functional mapping of rod physiology at cellular resolution. Successful endpoint of this project is to demonstrate the feasibility of super-resolution ophthalmoscopy of intact animals. In future phase of this project, the super-resolution ophthalmoscopy will be used to investigate biophysical mechanism of the TRP in normal animals, and to characterize abnormal modification of the TRP in transgenic animals with photoreceptor degeneration. Moreover, we also plan to combine adaptive optics with the proposed instrument to pursue super-resolution ophthalmoscopy of human subjects in following phase of this project.