McNulty Paul, Wu Rui, Yamaguchi Akihiro, Heckscher Ellie S, Haas Andrew, Nwankpa Amajindi, Skanata Mirna Mihovilovic, Gershow Marc
Department of Physics,New York University, New York, USA.
Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL.
bioRxiv. 2024 Dec 13:2024.05.22.595209. doi: 10.1101/2024.05.22.595209.
Direct measurement of neural activity in freely moving animals is essential for understanding how the brain controls and represents behaviors. Genetically encoded calcium indicators report neural activity as changes in fluorescence intensity, but brain motion confounds quantitative measurement of fluorescence. Translation, rotation, and deformation of the brain and the movements of intervening scattering or auto-fluorescent tissue all alter the amount of fluorescent light captured by a microscope. Compared to single-photon approaches, two photon microscopy is less sensitive to scattering and off-target fluorescence, but more sensitive to motion, and two photon imaging has always required anchoring the microscope to the brain. We developed a closed-loop resonant axial-scanning high-speed two photon (CRASH2p) microscope for real-time 3D motion correction in unrestrained animals, without implantation of reference markers. We complemented CRASH2p with a novel scanning strategy and a multi-stage registration pipeline. We performed volumetric ratiometrically corrected functional imaging in the CNS of freely moving larvae and discovered previously unknown neural correlates of behavior.
直接测量自由活动动物的神经活动对于理解大脑如何控制和表现行为至关重要。基因编码钙指示剂将神经活动报告为荧光强度的变化，但大脑运动混淆了荧光的定量测量。大脑的平移、旋转和变形以及中间散射或自发荧光组织的运动都会改变显微镜捕获的荧光量。与单光子方法相比，双光子显微镜对散射和离靶荧光不太敏感，但对运动更敏感，并且双光子成像一直需要将显微镜固定在大脑上。我们开发了一种闭环共振轴向扫描高速双光子（CRASH2p）显微镜，用于在无约束动物中进行实时3D运动校正，无需植入参考标记。我们用一种新颖的扫描策略和一个多阶段配准管道对CRASH2p进行了补充。我们在自由移动幼虫的中枢神经系统中进行了体积比率校正功能成像，并发现了以前未知的行为神经关联。
