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（3PM），用于神经元活动的大规模、非侵入性体积成像。为了利用基于绿色荧光蛋白的基因工程Ca探针的优异性能（例如，GCaMPs），将开发在1300 nm光谱窗口的3PM，其不仅在较长的激发波长下保持3PM的组织穿透能力，而且使各种各样的蓝色和绿色荧光团（包括许多荧光蛋白和Ca指示剂）能够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或更深的穿透深度下也可以实现用于成像动态脑活动的实际帧速率，将设计和制造新的物镜，以有效地收集深度处的信号。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AO成为神经科学实验室中的常规成像工具。为了提高SNR和空间分辨率，将采用1300 nm处的3PM中的AO。由于高阶非线性过程，AO对增加信号生成的影响对于3PM显著高于2PM。AO的影响预计也会随着成像深度的增加而增加。目标是在1300 nm处实现接近3PM衍射极限的空间分辨率，这将足以分辨单个枝晶。一个强大的跨学科研究团队已经组建，包括来自工业界和学术界的参与者，以执行研究和开发。该计划的成功完成将对神经科学产生广泛影响，因为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.