Optimization of 3-photon microscopy for Large Scale Recording in Mouse Brain

 描述(由申请人提供)：本研究计划的目标是优化三光子荧光显微镜(3 PM)，用于大规模、非侵入性、体积成像的神经元活动。为了利用基于绿色荧光蛋白的基因工程钙探针(例如GCaMPs)的卓越性能，将开发1300 nm光谱窗口的3 PM，这不仅保持了在较长激发波长下3 PM的组织穿透能力，而且使各种蓝色和绿色荧光团，包括一些荧光蛋白质和钙指示剂，可以通过三光子激发而被激发。为了提高信噪比(SNR)，以便即使在1.1 mm或更深的穿透深度也能获得实际的帧速率来成像动态脑活动，将设计和制造新的物镜，以便在深度有效地收集信号。此外，透镜设计还将支持与自适应光学(AO)的方便集成，目标是使自适应光学成为神经科学实验室的常规成像工具。为了同时提高信噪比和空间分辨率，将采用1300 nm的3 PM声光。由于高阶的非线性过程，声光对增加信号产生的影响在3 PM比2 PM显著更大。预计随着成像深度的增加，声光的影响也会增加。其目标是在1300 nm处获得接近衍射极限的3 PM的空间分辨率，这将足以分解单个树枝晶。已经组建了一支强大的跨学科研究团队，包括来自行业和学术界的参与者，以进行研究和开发。该项目的成功完成将对神经科学产生广泛的影响，神经科学需要在完整的小鼠大脑深处进行高分辨率、高速成像。

 DESCRIPTION (provided by applicant): The goal of this research program is to optimize three-photon fluorescence microscopy (3PM) for large scale, noninvasive, volumetric imaging of neuronal activity. To leverage the superb performance of green-fluorescent protein based genetically engineered Ca-probes (e.g., GCaMPs), 3PM at the 1300-nm spectral window will be developed, which not only preserves the tissue penetration capability of 3PM at the longer excitation wavelength but also enables a wide variety of blue and green fluorophores, including a number of fluorescent proteins and Ca-indicators, to be excitable via three-photon excitation. To improve the signal-to-noise ratio (SNR) so that a practical frame rate can be achieved for imaging dynamic brain activity even at a penetration depth of 1.1 mm or beyond, new objective lenses will be designed and fabricated that will collect the signal efficiently at depth. In additin, the lens design will also support convenient integration with adaptive optics (AO), with the goal of making AO a routine imaging tool in a neuroscience lab. To improve both SNR and spatial resolution, AO in 3PM at 1300 nm will be employed. The impact of AO for increasing signal generation is significantly higher for 3PM than 2PM because of the higher order nonlinear process. The impact of AO is also expected to increase with increasing imaging depth. The aim is to achieve close to diffraction limited spatial resolution for 3PM at 1300 nm, which will be sufficient to resolve individual dendrite. A strong interdisciplinary research team has been assembled, including participants from both industry and academia, to perform the research and development. The successful completion of this program will have a broad impact on neuroscience where high-resolution, high speed imaging deep within an intact mouse brain is required.