Transformative lightsheet microscopy techniques for subcellular imaging in physiologically relevant 3D environments

用于生理相关 3D 环境中亚细胞成像的变革性光片显微镜技术

• 批准号: 10712248
• 负责人: Tonmoy Chakraborty
• 金额: $ 35.72万
• 依托单位: UNIVERSITY OF NEW MEXICO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10712248
• 关键词: 3-Dimensional; Address; Architecture; Binding; Biological; Biological Process; Biology; Cell Communication; Cells; Characteristics; Classification; Color; Complex; Coupled; Coupling; Data; Development; Disease; Disseminated Malignant Neoplasm; Engineering; Environment; Event; Health; Image; Imaging Device; Intelligence; Label; Light; Lighting; Methods; Microscope; Microscopy; Molecular; Morphology; Optics; Organ; Organism; Organoids; Outcome; Pattern; Penetration; Phenotype; Physiological; Research; Research Personnel; Resolution; Sample Size; Sampling; Scanning; Specimen; Speed; Techniques; Technology; Time; Tissue imaging; Tissues; adaptive optics; cancer cell; cell type; design; fluorescence microscope; fluorophore; high resolution imaging; imaging modality; improved; interest; live cell imaging; millimeter; minimally invasive; next generation; novel; optical imaging; prevent; programs; spatiotemporal; temporal measurement; therapeutic target; treatment response; tumor microenvironment; user-friendly

Abstract Optical imaging enables fast and minimally invasive observation of biological processes within living cells and organisms. However, current state-of-the-art imaging instruments have limitations in acquisition speed, spatial resolution and light-penetration depth that restrict the types of biological questions that can be addressed. This is particularly problematic for biological samples that span several orders of magnitude in spatiotemporal scale. For example, cell-cell interactions within the tumor microenvironment and their response to treatment can occur over seconds to days and be heterogeneous throughout an entire tissue volume. Coupling these physiological outcomes to the underlying molecular mechanisms (and potential therapeutic targets) requires a transformation in not only the technologies we use, but also the combination of methods to cross the spatiotemporal scales from cells to tissues. Recent developments in emerging techniques like cleared-tissue-imaging coupled with lightsheet microscopy (LSM) has enabled researchers to probe deeper into the tissue without needing to section them. Illumination with lightsheet offers a much faster and less phototoxic alternative in comparison to point scanning microscopes. However, all LSM (including Lattice lightsheet) struggled with a number of fundamental limitations: (a) the maximum number of possible labels that can be imaged, (b) the size of the samples that they can handle, and, (c) poor spatial and temporal resolution. In order to fill these gaps my research program will engineer new optics that will not only improve the spatiotemporal resolution of the current state-of-the-art but also enable researchers to probe multiple simultaneous cellular phenotypes within the 3D architecture of the tissue microenvironment. By employing multiple scanning lightsheets we will develop a large volume hyperspectral LSM that will be able to unmix (segmentation and classification) 12+ fluorophores and image at 300 nm XYZ resolution to quantify the complex spatiotemporal interactions between various cell-types in tissue microenvironment. Additionally, we will develop a next generation LSM that will provide users a seamless transition from an organ/organism level imaging to 300 nm XYZ resolution. It will be proficient in identifying events-of-interest at lower resolution in large organs and intelligently adapt to high-resolution imaging, thus reducing imaging-time and generated-data burden. We will also design a new sample scanning strategy that will minimize light loss within the tissues. In order to prevent out-of-focus blur while imaging inside the tissue we will implement an autofocus routine that will enable users to carry out prolonged and unsupervised imaging of large specimens. Finally, we will develop a lattice lightsheet fluorescence microscope that will be able to perform fast, high-resolution multicolor imaging of live cells and spheroids. A configurable emission path will augment LSM with adaptive optics to counter sample induced aberrations. This will allow us to dynamically observe and quantify morphological phenotypes characteristic for highly metastatic cancer cells, which will be staged in organoids. I believe these have the potential to determine statistically significant patterns within the intact tissue that are bound to uncover novel biological questions.

摘要 光学成像能够快速和微创地观察活细胞内的生物过程和 有机体。然而，目前最先进的成像仪器在采集速度、空间分辨率等方面存在局限性。 以及光穿透深度，这限制了可以解决的生物学问题的类型。这是特别的 对于在时空尺度上跨越几个数量级的生物样本来说，这是一个问题。例如，cell-cell 肿瘤微环境内的相互作用及其对治疗的反应可能在几秒钟到几天内发生，并可能 在整个组织体积中是不均匀的。将这些生理结果与潜在的分子相结合 机制(和潜在的治疗靶点)不仅需要我们使用的技术的转变，而且还需要 从细胞到组织跨越时空尺度的方法组合。新兴市场的最新发展 像透明组织成像和光片显微镜(LSM)这样的技术使研究人员能够更深入地探索 进入组织而不需要切开它们。使用灯片照明可提供速度更快、光毒性更小的 与点扫描显微镜相比，它是另一种选择。然而，所有LSM(包括晶格LightSheet)都在与 基本限制的数量：(A)可以成像的可能标签的最大数量，(B) (C)空间和时间分辨率差。为了填补这些空白，我的研究项目 将设计新的光学技术，不仅将提高目前最先进的时空分辨率，而且还将 使研究人员能够在组织的3D架构内探索多种同时存在的细胞表型 微环境。通过使用多个扫描光板，我们将开发一种大体积高光谱LSM，它 将能够混合(分割和分类)12+荧光团和图像在300 nm XYZ分辨率来量化 组织微环境中不同细胞类型之间复杂的时空相互作用。此外，我们还将 开发新一代LSM，为用户提供从器官/生物体水平成像到300的无缝过渡 NM XYZ分辨率。它将擅长在较低分辨率的大型器官中识别感兴趣的事件和 智能适应高分辨率成像，从而减少成像时间和生成数据负担。我们还将设计 一种新的样本扫描策略，将组织内的光损失降至最低。为了防止失焦时的模糊 在组织内成像，我们将实施自动对焦程序，使用户能够进行延长和 大样本的非监督成像。最后，我们将开发一种晶格光片荧光显微镜，它将 能够对活细胞和球体进行快速、高分辨率的多色成像。可配置的排放路径将 用自适应光学技术增强LSM，以对抗样品引起的像差。这将使我们能够动态观察和 量化高转移性癌细胞的形态表型特征，这将在器官中分期。我 我相信这些有可能在完整的组织中确定统计上有意义的模式 发现新的生物学问题。