Multiphoton In Vivo Microscopy (Core 2)

多光子体内显微镜（核心 2）

基本信息

批准号：
10713243
负责人：
Kyle Patrick Lillis
金额：
$ 27.66万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
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：
2023-08-01 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10713243
关键词：
Algorithms Animals Biophysics Brain Calcium Calibration Chlorides Chronic Custom Data Data Collection Dedications Development Devices Dyes Epileptogenesis Experimental Designs Family suidae Fluorescence Fluorescence Microscopy Funding Image Imaging Device Injections Ions Measurement Measures Microscope Microscopic Microscopy Modeling Molecular Morphologic artifacts Motion Phase Post-Traumatic Epilepsy Procedures Research Resolution Resources Rodent Silicon Speed Technical Expertise Technology Traumatic Brain Injury Trephine hole adeno-associated viral vector cost cost effective data standards design digital extracellular flexibility fluorescence lifetime imaging fluorophore image archival system image registration imaging modality in vivo in vivo imaging in vivo two-photon imaging instrument instrumentation multi-photon multimodal data novel programs serial imaging tool tool development two-photon

项目摘要

Discovering the basic molecular and cellular mechanisms of post-traumatic epileptogenesis in a biophysically realistic model of traumatic brain injury will require having longitudinal, high-resolution access to the gyrencephalic, large animal brain. Fluorescence microscopy enhances such capabilities, enabling multimodal data collection through the use of genetically targeted and ion-selective fluorophores. In the tool development Phase I of this project, we developed an instrument and supporting technologies that afford us the unique ability to perform such measurements. These technologies include a custom two-photon microscope with a gantry design that can accommodate imaging in vivo, with subcellular resolution, brains of animals weighing >80kg. Microscopic in vivo imaging in animals of this size (to our knowledge, the largest by a factor of 2-4x) required substantial development and optimization of motion artifact mitigation tools, including heartbeat-triggered, high speed image stack acquisition; flexible, penetrable, transparent sub-dural windows; and post-hoc image registration algorithms. Finally, the microscope can perform digital fluorescence lifetime imaging, enabling quantification of extracellular chloride using novel single-wavelength dyes. The Microscopy Core will implement the imaging tools developed in Phase I, as well as optimize for calcium imaging and long-term repeated access. As these tools require substantial cost and technical expertise, the Microscopy Core represents a cost-effective consolidation and consistent implementation for the proposed program.

在A中发现创伤后癫痫发生的基本分子和细胞机制创伤性脑损伤的生物物理现实模型将需要纵向高分辨率进入大脑大脑的通心型。荧光显微镜增强了此类能力，通过使用遗传靶向和离子选择性来启用多模式数据收集荧光团。在该项目的工具开发第一阶段，我们开发了一种工具，支持我们执行此类测量的独特能力的支持技术。这些技术包括具有龙门设计的自定义两光子显微镜，可以容纳在体内成像，具有亚细胞分辨率，体重> 80kg的动物大脑。体内显微镜在这种大小的动物中成像（据我们所知，最大的2-4倍都需要大量运动伪像缓解工具的开发和优化，包括心跳触发，高速度图像堆栈采集；柔性，可穿透，透明的下窗；和事后图像注册算法。最后，显微镜可以执行数字荧光寿命成像，使用新型的单波长染料对细胞外氯化物进行定量。显微镜核心将实施在第一阶段开发的成像工具，并为钙成像和优化长期重复访问。由于这些工具需要大量的成本和技术专长，因此显微镜核心代表了成本效益的合并和一致的实施建议的计划。