High-throughput closed-loop direct aberration sensing and correction for multiphoton imaging in live animals

项目摘要 该项目的目标是提供实时像差校正的多光子成像和改进的信号比 细胞内研究活体动物混浊深层组织(~2 mm)的信噪比和空间分辨率 水平。多光子显微镜(MPM)因其高性能成为研究生物过程的一种有用工具 专属性和亚波长分辨率。特别是，与单光子成像相比，MPM使用激发 波长更长的光，可以更深地穿透组织，而非线性过程需要 产生三维局域激发的多光子相互作用。然而，高阶非线性 激发更容易受到焦点像差的影响，因此，在高散射情况下，穿透深度是有限的。 纸巾。自适应光学(AO)是一种很有前途的像差传感和矫正工具 系统。然而，现有的AO方法中的两个主要问题，1)精度和2)像差检测中的速度， 对活体深部组织实时成像仍然具有挑战性。 我建议开发一种新的用于MPM的高通量直接像差传感和校正方法， 称为共焦梯度光干涉显微镜(CGLIM)。这项技术旨在测量像差 使用共光路相移干涉仪消除系统和样品诱导的波前 像差，这反过来将改善激发焦点的质量，并增强信号强度。 具体地说，与其他努力相比，CGLIM使用的是长波长(~1.7μm)弹性背向散射光 从组织中直接测量激发光束的像差波前，导致大大降低 与荧光技术相比，消除光损伤、光漂白或热损伤的能力 生命系统的一部分。重要的是，CGLIM仅在焦平面附近测量像差波前 纳米级灵敏度(~2 nm或~0.002 rad)，这是由于它的公共路径，共聚焦配置。此外， CGLIM可以通过位相共轭自行验证像差传感的准确性。最后，像差 校正过程直接由CGLIM在闭环系统中的测量提供，无需任何迭代。CGLIM是 也容易在具有不同数字光圈的物镜的任何激光扫描系统中实现。 在提出的新方法中，我将首先演示使用CGLIM进行像差检测和校正 用组织模体和体外组织切片。然后，我将把CGLIM和三光子(3P)显微镜结合起来 活体小鼠和完整成年斑马鱼神经元活动的像差校正3P成像 大脑。最后，我的目标是将像差校正的MPM与自适应激励源和多边形相结合 内部开发的扫描技术用于研究活体大脑深层区域的实时神经元活动，而T细胞- 小鼠淋巴结深层区域的树突状细胞相互作用。 通过这个项目，我希望建立我对像差校正干涉术的研究重点 对活体动物的深层组织进行多光子成像，并使更多的神经科学和免疫学研究成为可能。

Project Summary This project aims to deliver real-time aberration-corrected multiphoton imaging with improved signal-to- noise-ratio (SNR) and spatial resolution for studying turbid deep-tissue (~2 mm) of living animals at the cellular level. Multiphoton microscopy (MPM) has been a useful tool to study biological processes due to its high specificity and sub-wavelength resolution. Particularly, compared to one-photon imaging, MPM uses excitation light with a longer wavelength that penetrates deeper into tissues, while the nonlinear process requires a multiphoton interaction that renders three-dimensional localized excitation. However, the higher-order nonlinear excitation is more susceptible to focus aberrations, thus, posing a limit for penetration depth in highly scattering tissues. Adaptive optics (AO) has been a promising tool for aberration sensing and correction for MPM in living systems. However, two major issues in existing AO methods, 1) accuracy, and 2) speed in aberration sensing, remain challenging to in vivo real-time deep-tissue imaging. I propose to develop a new high-throughput direct aberration sensing and correction method for MPM, termed confocal gradient light interference microscopy (CGLIM). This technique aims to measure the aberrated wavefront using a common-path, phase-shifting interferometer, to undo the systematic and specimen-induced aberration which, in turn, will improve the quality of the excitation focus and enhance the signal strength. Specifically, compared to other efforts, CGLIM uses the long-wavelength (~1.7 μm) elastic backscattered light from tissues to directly measure the aberrated wavefront of the excitation beam, resulting in substantially lower power compared to fluorescence techniques and eliminating photodamage, photobleaching, or heating damage of living systems. Importantly, CGLIM measures the aberrated wavefront only near the focal plane with nanoscale sensitivity (~2 nm or ~0.002 rad) owing to its common-path, confocal configuration. Furthermore, CGLIM can validate the accuracy of the aberration sensing by itself via phase conjugation. Lastly, the aberration correction procedure is directly fed by CGLIM’s measurement in a closed loop without any iterations. CGLIM is also readily implemented in any laser-scanning system with objectives of different numeric apertures. With the proposed new method, I will first demonstrate aberration sensing and correction using CGLIM with tissue phantoms and ex vivo tissue slices. Then, I will combine CGLIM with three-photon (3P) microscopy to demonstrate aberration-corrected 3P imaging of neuronal activities in live mice and intact adult zebrafish brains. Finally, I aim to combine the aberration-corrected MPM with the adaptive excitation source and polygon scanning developed in-house to study real-time neuronal activities in deep regions of live brains, and T cell – dendritic cell interactions in deep regions of a mouse’s lymph node. With this project, I hope to establish my research focus on aberration-corrected interferometric multiphoton imaging for deep tissues in living animals and enable more studies in neuroscience and immunology.