Expansion Optical Coherence Microscopy (ExOCM)

扩展光学相干显微镜 (ExOCM)

• 批准号: 10668523
• 负责人: Chao Zhou
• 金额: $ 19.46万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10668523
• 关键词: 3-Dimensional; Adopted; Adoption; Biomedical Research; Blood Vessels; Cell Communication; Cell Differentiation process; Cells; Color; Communities; Contrast Media; Data; Developmental Biology; Diameter; Dimensions; Dyes; Fluorescence; Growth; Hydrogels; Image; Imaging Device; Label; Light; Microscope; Microscopy; Modeling; Molecular; Molecular Profiling; Optical Coherence Tomography; Optics; Organoids; Process; Proteins; Protocols documentation; Regenerative Medicine; Research; Resolution; Sampling; Scanning; Signal Transduction; Specimen; Speed; Structure; System; Techniques; Technology; Thick; Three-Dimensional Image; Time; Tissue Expansion; Tissue Sample; Tissues; Tumor Angiogenesis; Water; absorption; angiogenesis; antibody conjugate; anticancer research; cell type; detection sensitivity; experience; experimental study; fluorescence microscope; imaging approach; imaging capabilities; imaging platform; imaging system; improved; instrument; interest; meter; microscopic imaging; multiplexed imaging; nanoGold; nanometer resolution; new technology; organ growth; programs; structural imaging; superresolution imaging; technology development; tool; tumor; two photon microscopy; two-photon; ultra high resolution

Project Summary Inspired by recent advances in expansion microscopy (ExM), we propose to develop a new technology called expansion optical coherence microscopy (ExOCM). Using a standard optical coherence tomography (OCT) or a high-resolution OCM system, we will obtain super-resolution, molecular-specific images of expanded tissue samples. Our recent pilot experiment demonstrated that intrinsic scattering signals from expanded samples can be observed using OCT, demonstrating the feasibility of ExOCM based on intrinsic scattering contrast. To further enable molecular-specific imaging, contrast agents, such as gold nanoparticles (GNPs), molecular dyes, and even fluorescence proteins, with different light absorption profiles can be selected for multi-color ExOCM. Compared to standard ExM, ExOCM can image extra thick samples at high speed. This approach also allows the co-registration of three-dimensional (3D) molecular profiles with live tissue microstructures obtained from the same specimen using label-free OCT. Building upon our group’s extensive expertise in developing OCT and OCM technologies and experience with 3D organoid models, we propose to develop, optimize and validate ExOCM technology, and demonstrate the unique attributes of ExOCM for biomedical research. Two specific aims of the proposed program are: Aim 1. To develop and optimize ExOCM technology for multi-color, super- resolution imaging of expanded organoids. We will optimize an ultrahigh-resolution OCM system to achieve ~1.5 µm isotropic image resolution. Antibody conjugated GNPs will be used to provide selective targeting for specific proteins. Using our optimized OCM system and a ~4.5X tissue expansion protocol, we expect to obtain a ~300 nm resolution in both the axial and transverse dimensions. Multi-color and multiplexed imaging will be obtained using spectroscopic OCT analysis to separate signals from GNPs with different absorption and scattering profiles. Aim 2. To validate ExOCM with two-photon microscopy, and to demonstrate the feasibility of the combined OCT and ExOCM imaging approach to characterize 3D structural and molecular profiles and growth dynamics from the same samples. We will use OCT to acquire longitudinal, label-free images of living organoids over a three-week time-course. At predetermined time points, samples will be expanded for ExOCM and two- photon imaging, where tissue structural landmarks will be identified from 3D image stacks for registration. ExOCM imaging data collected during the expansion process will provide first-hand information about 3D expansion uniformity. Complementary 3D structural and molecular profiles and growth dynamics from the same organoids will be obtained, demonstrating the unique advantages of the combined OCT and ExOCM imaging approach. If successful, the integrated OCT and ExOCM imaging platform will prove helpful for applications in developmental biology, cancer research and regenerative medicine. Furthermore, high-throughput characterization of the specimens can be performed using commercially available OCT systems, making ExOCM easy to disseminate and adopt by the broader research community.

项目概要 受膨胀显微镜（ExM）最新进展的启发，我们建议开发一种称为 扩展光学相干显微镜（ExOCM）。使用标准光学相干断层扫描 (OCT) 或 高分辨率 OCM 系统，我们将获得扩张组织的超分辨率、分子特异性图像 样品。我们最近的试点实验表明，来自扩展样品的固有散射信号可以 使用 OCT 进行观察，证明了基于固有散射对比度的 ExOCM 的可行性。为了进一步 实现分子特异性成像、造影剂，例如金纳米颗粒 (GNP)、分子染料和 甚至可以选择具有不同光吸收曲线的荧光蛋白用于多色 ExOCM。 与标准 ExM 相比，ExOCM 可以高速对超厚样品进行成像。这种方法还允许 三维 (3D) 分子轮廓与从活组织获得的微观结构的共同配准 使用无标记 OCT 的同一样本。凭借我们团队在开发 OCT 和 OCM 技术和 3D 类器官模型经验，我们建议开发、优化和验证 ExOCM技术，并展示了ExOCM对于生物医学研究的独特属性。两个具体 拟议计划的目标是： 目标 1. 开发和优化 ExOCM 技术，用于多色、超 扩展类器官的分辨率成像。我们将优化超高分辨率 OCM 系统以达到 ~1.5 µm 各向同性图像分辨率。抗体偶联的 GNP 将用于为特定的目标提供选择性靶向 蛋白质。使用我们优化的 OCM 系统和 ~4.5X 组织扩张方案，我们预计获得 ~300 轴向和横向尺寸的纳米分辨率。将获得多色和多重成像 使用光谱 OCT 分析从具有不同吸收和散射的 GNP 中分离信号 配置文件。目标 2. 用双光子显微镜验证 ExOCM，并证明该方法的可行性 结合 OCT 和 ExOCM 成像方法来表征 3D 结构和分子概况以及生长 来自相同样本的动力学。我们将使用 OCT 获取活体类器官的纵向、无标记图像 为期三周的课程。在预定的时间点，样本将被扩展为 ExOCM 和两个- 光子成像，将从 3D 图像堆栈中识别组织结构标志进行配准。 扩建过程中收集的ExOCM成像数据将提供有关3D的第一手资料 膨胀均匀性。互补的 3D 结构和分子概况以及生长动态 将获得类器官，展示 OCT 和 ExOCM 成像相结合的独特优势 方法。如果成功，集成的 OCT 和 ExOCM 成像平台将有助于以下领域的应用： 发育生物学、癌症研究和再生医学。此外，高通量 可以使用市售 OCT 系统对样本进行表征，从而使 ExOCM 易于传播并被更广泛的研究界采用。