Optimization of multiphoton microscopy for large scale activity mapping in adult zebrafish

脊椎动物的行为来自于大脑中神经元的相互作用，但揭示这些行为的工具 活体动物的细胞水平上的神经元结构和功能只能进入大脑的一小部分。我们 必须朝着接近个体成年行为动物大脑中任何地方的结构和功能的方向发展。 体内三光子（3 P）显微镜，最近，但证明，技术允许光学访问更深 结构比以往任何时候都在完整的哺乳动物大脑，但仍有许多优化，以催化其更广泛的 领养该项目的计划是扩大3 P显微镜的范围，包括大脑内部和整个大脑。 科学界通过开发新技术并证明其对成像结构和功能的价值 在成年斑马鱼大脑的任何地方-一个强大的脊椎动物模型。该项目将扩大深度， 通过技术改进的组合，2 P和3 P成像的速度和区域范围。 通过发展一种新的双自适应光学方法来校正光学误差， 结合共轭和标准自适应光学系统的像差， 接近衍射极限的分辨率和改善的信号。为了提高成像的广度和速度， 将开发一种重量轻、体积小、串联压电纤维扫描引擎和大视场的方法 具有光栅扫描场中任何2到4个子区域的能力。通过确定并应用最佳 激光重复率和非线性激发的最佳阶数作为深度的函数， 神经元将增加，可以成像，减少光暴露，以改善长期，重复 生活中的影像虽然这些创新将对许多动物模型有用，但它们将通过以下方法进行测试： 利用CRISPR技术对新生成的转基因斑马鱼品系进行成像， 建立的线路。这些线标记具有膜靶向的不同递质表型的神经元 用于结构成像的荧光团，或用于功能成像的遗传编码钙指示剂（gCaMPf或s）。 显像生物学前沿的目标是提供一种工具， 在完整的个体脊椎动物的大脑中的任何地方的结构和功能，在其生命的任何时间，从 胚胎进入成年期。

Vertebrate behaviors emerge from interactions of neurons across the brain, but the tools for revealing neuronal structure and function at the cellular level in living animals access only small portions of the brain. We must move toward access to structure and function anywhere in the brain of individual adult, behaving animals. In vivo three photon (3P) microscopy, a recent, but proven, technology allows optical access to deeper structures than ever before in intact mammalian brains, but much optimization remains to catalyze its wider adoption. The plan of this project is to extend the reach of 3P microscopy both within brains and through the scientific community by developing new technology and proving its worth for imaging structure and function anywhere in the brain of adult zebrafish – a powerful vertebrate model. This project will extend the depth, speed and regional extent of imaging with 2P and 3P through a combination of technological improvements. Imaging depth will be enhanced by the development of a novel dual adaptive optics approach to correct optical aberrations that combines conjugate and standard adaptive optics to allow deep imaging through the skull with near diffraction limited resolution and improved signal. To enhance the breadth and speed of imaging, a novel approach will be developed with a light-weight, small, tandem piezo-fiber scan engine and a large field of view with the ability to raster scan any 2 to 4 sub-regions in the field. By determining and then applying optimum laser repetition rates and the best order of the nonlinear excitation as a function of depth, the number of neurons will be increased that can be imaged and reduce light exposure to improve longer term, repeated imaging through life. While the innovations will be useful for many animal models, they will be tested by imaging newly generated transgenic zebrafish lines made with CRISPR technology, as well as other established lines. The lines label neurons of different transmitter phenotype with membrane targeted fluorophores for structural imaging, or genetically encoded calcium indicators (gCaMPf or s) for functional imaging. The goal on the biological front is to provide the tools to allow the unique ability to image neuronal structure and function anywhere in the brain of an intact individual vertebrate at any time during its life, from embryo into adulthood.