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

用于活体动物多光子成像的高通量闭环直接像差传感和校正

• 批准号: 10572572
• 负责人: Xi Chen
• 金额: $ 10.41万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10572572
• 关键词: 3-Dimensional; Acute; Adult; Animals; Attenuated; Behavior; Biological Process; Biomedical Research; Brain; Cancer Biology; Cell Communication; Complex; Dendritic Cells; Deterioration; Elasticity; Fluorescence; Foundations; Goals; Heating; Image; Imaging Device; Immune response; Immunology; Interference Microscopy; Lasers; Light; Lighting; Measurement; Measures; Methods; Microscope; Microscopy; Mus; Muscle; Neurons; Neurosciences; Noise; Operative Surgical Procedures; Optical Methods; Organism; Penetration; Phase; Photobleaching; Photons; Predisposition; Procedures; Process; Research; Resected; Resolution; Scanning; Scientist; Signal Transduction; Slice; Source; Specificity; Specimen; Speed; System; T-Lymphocyte; Techniques; Technology; Thalamic structure; Time; Tissue Sample; Tissue imaging; Tissues; Tube; Zebrafish; adaptive optics; career; cell motility; cellular imaging; confocal imaging; fluorophore; improved; in vivo; lymph nodes; multi-photon; multiphoton imaging; multiphoton microscopy; nanoscale; new technology; non-invasive system; novel; photomultiplier; programs; reconstruction; submicron; three photon microscopy; tissue phantom; tool; two-photon

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.

项目摘要 该项目旨在提供实时畸变校正的多光子成像，并改善信号对 - 噪声比率（SNR）和空间分辨率用于研究细胞处的活动物浊度（〜2 mm） 等级。多光子显微镜（MPM）是研究生物学过程的有用工具 特异性和次波长分辨率。特别是，与单光子成像相比，MPM使用兴奋 光线具有更长的波长，可深入组织，而非线性过程需要 多光子相互作用，它具有三维局部兴奋。但是，高阶非线性 兴奋更容易焦点畸变，因此，在高度散射中构成了渗透深度的极限 自适应光学元件（AO）一直是MPM在生活中的畸变感应和校正的有前途的工具 系统。但是，现有AO方法中的两个主要问题，1）准确性和2）速度的差速感应， 对体内实时深度组织成像保持挑战。 我建议为MPM开发一种新的高通量直接畸变感应和校正方法， 称为共聚焦梯度光干扰显微镜（CGLIM）。该技术旨在测量畸形 波前使用通用路径，相位转移干涉仪撤消系统和标本诱导的 畸变反过来将提高兴奋聚焦的质量并增强信号强度。 具体而言，与其他努力相比，CGLIM使用长波长（〜1.7μm）弹性反向散射光 从组织到直接测量兴奋束的异常波前，导致较低 与荧光技术相比，电力并消除了光损伤，光漂白或加热损害 生活系统。重要的是，CGLIM仅在焦平面附近测量了一个畸变的波前 纳米级敏感性（〜2 nm或〜0.002 rad），由于其共同路径的共聚焦配置。此外， CGLIM可以通过相结合本身验证像差传感的准确性。最后，畸变 CGLIM在封闭环中的测量直接喂养校正程序，而无需任何迭代。 CGLIM是 也很容易在任何激光扫描系统中实现，具有不同的数字孔径的目标。 使用拟议的新方法，我首先使用CGLIM证明异常敏感性和校正 带有组织幻像和离体组织切片。然后，我将CGLIM与三光子（3p）显微镜相结合 展示活小鼠中神经元活性的像差校正的3P成像和完整的成年斑马鱼 大脑。最后，我的目标是将被像差校正的MPM与自适应兴奋源和多边形结合 扫描开发了内部，以研究活体大脑和T细胞深处的实时神经元活动 - 小鼠淋巴结深区域中的树突状细胞相互作用。 在这个项目中，我希望将我的研究重点放在被像差校正的干涉量学上 对活动物中深层组织的多光子成像，并在神经科学和免疫学方面进行了更多研究。