High Resolution Functional Imaging of the Retina

项目摘要 需要非侵入性的方法来可视化单个细胞和评估分子动力学 以提高我们对活体眼睛的视觉功能和细胞代谢的了解。其中一些 参与视觉周期和细胞能量产生的关键分子本质上是荧光的。 然而，它们在使用单光子激发的活体眼睛中是无法获得的，因为激发 波长范围不是由眼睛的光学系统传输的。我们已经研制出双光子激发 荧光眼底检查(TPEFO)，以激发这些本来无法接近的荧光团与近 红外光与自适应光学扫描光检眼镜相结合，使非 在活体视网膜中对许多不同细胞类别的侵入性高分辨率成像。使用 TPEFO，视觉循环动力学可以通过发射双光子的强度变化来表征 来自光感受器的激发荧光。在此资助期间，TPEFO的独特能力 将得到增强，以在临床前模型中测量额外的有意义的荧光特性。AS 一个了解分子动力学视觉循环的窗口，我们将结合动力学来跟踪 对视觉刺激所发出的荧光的响应，荧光寿命的变化 光感受器。我们的第二个目标是通过跟踪发射信号来非侵入性地测量神经元功能。 来自视网膜内部的双光子激发荧光动力学和寿命。最后，我们将比较 在不同光谱波段发射的TPEFO强度，以计算光学氧化还原比，这是一种测量 细胞在有氧和无氧代谢之间的平衡。所有这些措施都可能是 细胞健康和功能。除了在健康眼睛中表征TPEFO的特性外，我们还将使用 全身性低氧作为细胞呼吸改变的模型来代表发生的潜在变化 在疾病的早期。这项研究有可能提供对正常和改变的生物化学的洞察。 并提高我们对影响视网膜代谢和视力的疾病的理解 青光眼、黄斑变性和Leber遗传性视神经病变等功能。

Project Summary Non-invasive methodologies to visualize individual cells and assess molecular dynamics are needed to improve our understanding of visual function and cellular metabolism in the living eye. Some of the key molecules involved in the visual cycle and cellular energy production are intrinsically fluorescent. However, they are inaccessible in the living eye using single-photon excitation because the excitation wavelength range is not transmitted by the optics of the eye. We have developed two-photon excited fluorescence ophthalmoscopy (TPEFO) to excite these otherwise inaccessible fluorophores with near- infrared light in conjunction with an adaptive optics scanning light ophthalmoscope that enables non- invasive high-resolution imaging of many different cell classes throughout the in vivo retina. With TPEFO, visual cycle kinetics can be characterized through the intensity changes of emitted two-photon excited fluorescence from photoreceptors. During this funding period, the unique capabilities of TPEFO will be enhanced to measure additional meaningful fluorescence properties in pre-clinical models. As a window into the molecular dynamics of the visual cycle, we will track, in conjunction with the kinetics of emitted fluorescence in response to visual stimulation, changes in the fluorescence lifetime of photoreceptors. Our second aim is to non-invasively measure neuronal function by tracking emitted two-photon excited fluorescence kinetics and lifetime from the inner retina. Finally, we will compare the TPEFO intensity emitted in different spectral bands to calculate the optical redox ratio, a measure of a cell's balance between aerobic and anaerobic metabolism. All of these measures may be indicators of cell health and function. In addition to characterizing the TPEFO properties in healthy eyes, we will use systemic hypoxia as a model of altered cellular respiration to represent the potential changes that occur early in disease. This research has the potential to provide insight into normal and altered biochemical processes and improve our understanding of diseases that impact retinal metabolism and visual function such as glaucoma, macular degeneration and Leber hereditary optic neuropathy.