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.

抽象的 光学成像能够快速、微创地观察活细胞内的生物过程， 有机体。然而，当前最先进的成像仪器在采集速度、空间分辨率方面存在局限性 和光穿透深度限制了可以解决的生物学问题的类型。这一点特别 对于时空尺度跨越几个数量级的生物样本来说是有问题的。例如，细胞-细胞 肿瘤微环境内的相互作用及其对治疗的反应可能会在几秒到几天内发生，并且 整个组织体积中的异质性。将这些生理结果与潜在的分子耦合 机制（和潜在的治疗靶点）不仅需要我们使用的技术发生转变，而且还需要改变 跨时空尺度从细胞到组织的方法组合。新兴市场的最新发展 透明组织成像与光片显微镜 (LSM) 相结合等技术使研究人员能够进行更深入的探索 无需将其切片即可进入组织。使用 Lightsheet 进行照明可提供更快且光毒性更小的效果 与点扫描显微镜相比的替代方案。然而，所有 LSM（包括 Lattice lightsheet）都面临着一个问题： 基本限制的数量：（a）可以成像的可能标签的最大数量，（b）标签的大小 他们可以处理的样本，以及（c）空间和时间分辨率差。为了填补这些空白我的研究计划 将设计新的光学器件，不仅提高当前最先进技术的时空分辨率，而且 使研究人员能够同时探测组织 3D 结构内的多个细胞表型 微环境。通过采用多个扫描光片，我们将开发一种大体积高光谱 LSM 将能够分解（分割和分类）12+荧光团并以 300 nm XYZ 分辨率成像以进行定量 组织微环境中各种细胞类型之间复杂的时空相互作用。此外，我们将 开发下一代 LSM，为用户提供从器官/生物体水平成像到 300 级成像的无缝过渡 纳米 XYZ 分辨率。它将熟练地识别大型器官中较低分辨率的感兴趣事件 智能地适应高分辨率成像，从而减少成像时间和生成的数据负担。我们也会设计 一种新的样本扫描策略，可最大限度地减少组织内的光损失。为了防止失焦模糊 我们将实施自动对焦程序，使用户能够在组织内部进行长时间和连续的成像 大样本的无监督成像。最后，我们将开发一种晶格光片荧光显微镜 能够对活细胞和球体进行快速、高分辨率的多色成像。可配置的发射路径将 使用自适应光学增强 LSM，以抵消样本引起的像差。这将使我们能够动态地观察和 量化高度转移癌细胞的形态表型特征，这些细胞将在类器官中进行分期。我 相信这些有可能确定完整组织内具有统计学意义的模式，这些模式必然与 发现新的生物学问题。