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.

项目概要 该项目旨在提供实时像差校正多光子成像，并改进信号到 噪声比 (SNR) 和空间分辨率，用于在细胞中研究活体动物的浑浊深层组织 (~2 mm) 多光子显微镜（MPM）因其高水平而成为研究生物过程的有用工具。 特别是，与单光子成像相比，MPM 使用激发。 波长较长的光可以更深入地穿透组织，而非线性过程需要 多光子相互作用呈现三维局部激发然而，高阶非线性。 激发更容易受到焦点像差的影响，因此，在高散射中限制了穿透深度 自适应光学 (AO) 已成为活体 MPM 的像差传感和校正的有前途的工具。 然而，现有 AO 方法存在两个主要问题，1）精度，2）像差传感的速度， 体内实时深层组织成像仍然具有挑战性。 我建议为 MPM 开发一种新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），该技术旨在测量像差。 使用共路径相移干涉仪进行波前处理，以消除系统和样本引起的干扰 像差，反过来又会提高激励焦点的质量并增强信号强度。 具体来说，与其他研究相比，CGLIM 使用长波长（~1.7 μm）弹性背散射光 从组织中直接测量激发光束的像差波前，从而显着降低 与荧光技术相比功率更高，并消除光损伤、光漂白或热损伤 重要的是，CGLIM 仅测量焦平面附近的像差波前。 由于其共路径、共焦配置，纳米级灵敏度（~2 nm 或~0.002 rad）。 CGLIM 可以通过相位共轭来验证像差传感的准确性。 校正过程直接由闭环中的 CGLIM 测量提供，无需任何迭代。 也很容易在具有不同数值孔径物镜的任何激光扫描系统中实现。 通过所提出的新方法，我将首先使用 CGLIM 演示像差传感和校正 然后，我将 CGLIM 与三光子 (3P) 显微镜结合起来。 演示活体小鼠和完整成年斑马鱼神经活动的像差校正 3P 成像 最后，我的目标是将像差校正 MPM 与自适应激励源和多边形结合起来。 内部开发的扫描技术用于研究活体大脑深层区域和 T 细胞的实时神经活动 – 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，我希望将我的研究重点放在像差校正干涉测量上 用于活体动物深层组织的多光子成像，使更多的神经科学和免疫学研究成为可能。