Super-resolved multiphoton microscopy with dual output ultrafast laser

项目总结/摘要 神经系统疾病可能对认知、情感或感觉运动功能产生破坏性影响。基因 使用腺相关病毒（AAV）载体的递送已经证明了治疗 影响中枢和周围神经系统的先天性和后天性疾病。推进 对神经系统细胞内载体摄取和运输的机制的理解将告知病毒 载体衣壳设计。因此，体内病毒载体细胞转导的可视化受到以下因素的阻碍： 缺乏解决厚组织中纳米级颗粒的最佳方法。成像病毒颗粒， 光学显微镜的衍射极限分辨率通常使用 电子显微镜，一种与生命不相容的资源密集型技术。迫切需要发展 活体成像技术，能够对生命系统进行高速和深度纳米级成像。二... 光子激发（2 PE）显微术是用于神经系统活体成像的强大技术， 采用能够深入穿透组织的超快近红外激光。尽管技术 能够对厚组织进行活体成像，2 PE显微镜可达到的分辨率和图像质量 不足以研究纳米工艺。2 PE显微镜可以与受激发射配对 耗尽（STED）技术，使纳米级荧光标记的目标的分辨率。加强 成像深度和信号-背景，2 PE可以通过两个超快的时空重叠来实现。 在称为非简并2 PE的过程中使用不同波长的激光器。因此，广泛传播 2 PE-STED和非简并2 PE显微镜技术受到成本和复杂性的阻碍 与两个超快激光源的同步和对准相关。在此，我们建议开发 增强时空分辨率和成像的高效超分辨多光子显微镜方法 通过采用单个双输出商用超快激光器，在活组织内进行深度测量。一旦开发出来，我们 将使用这些新的成像平台来研究单个AAV颗粒在细胞内的细胞内运输， 老鼠的神经系统在目标1中，将利用双输出超快激光实现2 PE-STED 通过脉冲耗尽的显微镜，并用于对培养的雪旺细胞和原代细胞中的AAV运输进行成像。 小鼠背根神经节感觉神经元。在目标2中，双输出超快激光器将用于 实现非简并2 PE，并与连续波耗尽光束配对，用于深度超分辨率 在活的麻醉小鼠中，在角膜雪旺细胞和感觉神经元中的AAV运输的成像。液体 将采用透镜来提高体积成像速度。如果成功，这项工作将有可能 通过识别细胞内运输来进一步理解神经系统细胞的病毒载体转导 瓶颈，同时说明了超分辨率2 PE显微镜技术的潜力，以推进 生物医学知识发现

Project Summary/ Abstract Nervous system disease may yield devastating impact on cognition, emotion, or sensorimotor function. Gene delivery using adeno-associated virus (AAV) vectors has demonstrated immense potential for treatment of congenital and acquired diseases impacting the central and peripheral nervous systems. Advancing mechanistic understanding of vector uptake and trafficking within nervous system cells would inform viral vector capsid design. Heretofore, visualizing viral vector cellular transduction in vivo has been hampered by a lack of optimal means for resolving nanoscale particles in thick tissues. Imaging viral particles whose dimensions are below the ~250 nm diffraction limit resolution of light microscopy is typically achieved using electron microscopy, a resource-intensive technique incompatible with life. There is a critical need to develop intravital imaging techniques that enable high-speed and deep nanoscale imaging of living systems. Two- photon excitation (2PE) microscopy is a powerful technique for intravital imaging of the nervous system that employs ultrafast near-infrared laser light capable of penetrating deep into tissues. Though the technique enables intravital imaging of thick tissues, the achievable resolution and image quality of 2PE microscopy is inadequate for study of nanoscale processes. 2PE microscopy may be paired with stimulated emission depletion (STED) techniques to enable resolution of nanoscale fluorescent-tagged targets. To enhance imaging depth and signal-to-background, 2PE may be achieved via spatiotemporal overlap of two ultrafast lasers of different wavelengths in a process termed non-degenerate 2PE. Heretofore, wide dissemination of 2PE-STED and non-degenerate 2PE microscopy techniques have been hampered by the cost and complexity associated with synchronization and alignment of two ultrafast laser sources. Herein, we propose to develop efficient super-resolved multiphoton microscopy approaches to enhance spatiotemporal resolution and imaging depth within living tissues by employing a single dual-output commercial ultrafast laser. Once developed, we will employ these novel imaging platforms to study intracellular trafficking of single AAV particles within cells of the murine nervous system. In Aim 1, the dual-output ultrafast laser will be utilized to achieve 2PE-STED microscopy via pulsed depletion and employed to image AAV trafficking in cultured Schwann cells and primary sensory neurons of murine dorsal root ganglia. In Aim 2, the dual-output ultrafast laser will be utilized to achieve non-degenerate 2PE and paired with a continuous wave depletion beam for deep super-resolution imaging of AAV trafficking in corneal Schwann cells and sensory neurons in live anesthetized mice. A liquid lens will be employed to enhance volumetric imaging speed. If successful, this work carries potential to advance understanding of viral vector transduction of nervous system cells by identifying intracellular trafficking bottlenecks, while illustrating the potential of super-resolution 2PE microscopy techniques to advance knowledge discovery in biomedicine.