Super-resolved multiphoton microscopy with dual output ultrafast laser

具有双输出超快激光的超分辨率多光子显微镜

基本信息

批准号：
10664267
负责人：
Ivan Coto Hernandez
金额：
$ 14.28万
依托单位：
MASSACHUSETTS EYE AND EAR INFIRMARY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-01 至 2026-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10664267
关键词：
3-Dimensional Acoustics Afferent Neurons Anesthesia procedures Animal Model Axon Back Biomedical Research Biotechnology Capsid Cells Central Nervous System Cognition Complex Computer software Cornea Dependovirus Development Dimensions Disease Drug Design Dyes Electron Microscopy Emotions Engineering Fiber Gene Delivery Goals Image Image Enhancement Imaging Device Imaging Techniques In Vitro Knowledge Discovery Label Lasers Life Light Liquid substance Mentors Microscope Microscopy Mus Nervous System Neurons Nuclear Translocation Optics Organism Output Pattern Penetration Peripheral Nervous System Physics Physiologic pulse Positioning Attribute Process Resolution Resources Schwann Cells Sensorimotor functions Sensory Shapes Signal Transduction Source Specimen Speed Spinal Ganglia Stretching Structure Techniques Therapeutic Thick Time Tissues Venus Viral Viral Vector Virion Visualization Work adeno-associated viral vector biomedical imaging cell type cellular transduction cost design expectation imaging platform improved in vivo insight intravital imaging lens light microscopy multi-photon multiphoton microscopy nanoscale nervous system disorder nervous system imaging novel open source particle permissiveness skills spatiotemporal superresolution imaging trafficking two-photon ultra high resolution uptake vector virology

项目摘要

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.

项目概要/摘要神经系统疾病可能会对认知、情绪或感觉运动功能产生毁灭性影响。基因使用腺相关病毒（AAV）载体进行递送已证明治疗以下疾病的巨大潜力影响中枢和周围神经系统的先天性和后天性疾病。前进对神经系统细胞内载体摄取和运输的机制的理解将为病毒提供信息矢量衣壳设计。迄今为止，体内病毒载体细胞转导的可视化一直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。对病毒颗粒进行成像尺寸低于光学显微镜的〜250 nm衍射极限分辨率通常是使用电子显微镜是一种与生命不相容的资源密集型技术。迫切需要开发能够对生命系统进行高速、深度纳米级成像的活体成像技术。二- 光子激发 (2PE) 显微镜是一种用于神经系统活体成像的强大技术，采用能够深入组织的超快近红外激光。虽然技术能够对厚组织进行活体成像，2PE 显微镜可实现的分辨率和图像质量是不足以研究纳米级过程。 2PE 显微镜可与受激发射配合使用耗尽（STED）技术可实现纳米级荧光标记目标的分辨率。增强成像深度和信号到背景，2PE 可以通过两个超快的时空重叠来实现在称为非简并 2PE 的过程中使用不同波长的激光。迄今为止，广泛传播 2PE-STED 和非简并 2PE 显微镜技术因成本和复杂性而受到阻碍与两个超快激光源的同步和对准相关。在此，我们建议开发有效的超分辨率多光子显微镜方法可增强时空分辨率和成像通过使用单个双输出商用超快激光器来测量活体组织内的深度。一旦开发出来，我们将利用这些新颖的成像平台来研究单个 AAV 颗粒在细胞内的细胞内运输小鼠神经系统。在目标1中，将利用双输出超快激光器来实现2PE-STED 通过脉冲耗尽显微镜，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的感觉神经元。在目标 2 中，双输出超快激光器将用于实现非简并 2PE 并与连续波耗尽光束配对以实现深度超分辨率活体麻醉小鼠角膜雪旺细胞和感觉神经元中 AAV 运输的成像。液体将采用透镜来提高体积成像速度。如果成功的话，这项工作有潜力通过识别细胞内运输促进对神经系统细胞病毒载体转导的理解瓶颈，同时说明超分辨率 2PE 显微镜技术的潜力生物医学知识发现。