Expansion Optical Coherence Microscopy (ExOCM)

扩展光学相干显微镜 (ExOCM)

• 批准号: 10530971
• 负责人: Chao Zhou
• 金额: $ 23.63万
• 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 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10530971
• 关键词: 3-Dimensional; Adopted; Adoption; Biomedical Research; Blood Vessels; Caliber; Cell Communication; Cell Differentiation process; Cells; Color; Communities; Contrast Media; Data; Developmental Biology; 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; System; Techniques; Technology; Thick; Three-Dimensional Image; Time; Tissue Expansion; Tissue Sample; Tissues; Tumor Angiogenesis; Water; absorption; angiogenesis; antibody conjugate; anticancer research; base; cell type; detection sensitivity; experience; experimental study; fluorescence microscope; imaging approach; imaging capabilities; imaging platform; imaging system; improved; instrument; interest; microscopic imaging; multiplexed imaging; nanoGold; nanometer resolution; new technology; organ growth; programs; technology development; tool; tumor; two photon microscopy; two-photon

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.

项目摘要 受膨胀显微镜(Exm)最新进展的启发，我们建议开发一种新技术，称为 膨胀光学相干显微镜(ExOCM)。使用标准光学相干层析成像(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 样本。我们最近的试点实验表明，来自扩展样本的本征散射信号可以 利用OCT进行了观测，证明了基于本征散射对比度的ExOCM的可行性。为了进一步 启用分子特定成像、造影剂，如金纳米颗粒(GNPs)、分子染料和 甚至具有不同光吸收谱的荧光蛋白也可以被选择用于多颜色ExOCM。 与标准EXM相比，ExOCM可以高速成像超厚样品。这种方法还允许 将三维(3D)分子轮廓与来自 同样的样本使用无标签OCT。基于集团在开发OCT和OCT方面的广泛专业知识 OCM技术和3D有机物模型的经验，我们建议开发、优化和验证 ExOCM技术，并展示了ExOCM在生物医学研究中的独特属性。两个具体的 该方案的目标是：目标1.开发和优化ExOCM技术，以实现多色、超级 膨胀有机物的分辨率成像。我们将优化一个超高分辨率的OCM系统，使其达到~1.5 各向同性图像分辨率。抗体结合的GNPs将被用来提供选择性靶向特定的 蛋白质。使用我们优化的OCM系统和~4.5倍的组织扩张方案，我们预计将获得~300 轴向和横向两个维度的NM分辨率。将获得多色和多路复用的成像 利用OCT光谱分析从不同吸收和散射的GNPs中分离信号 配置文件。目的2.用双光子显微镜验证ExOCM，并论证其可行性。 OCT和ExOCM联合成像方法表征三维结构和分子轮廓及生长 来自相同样本的动力学数据。我们将使用OCT来获取纵向的、无标签的活体图像 为期三周的课程。在预定的时间点，样本将扩展为ExOCM和两个- 光子成像，其中组织结构地标将从3D图像堆栈中识别出来进行配准。 扩展过程中收集的ExOCM成像数据将提供有关3D的第一手信息 膨胀均匀度。互补的3D结构和分子轮廓以及来自其的生长动力学 将获得有机化合物，展示了OCT和ExOCM联合成像的独特优势 接近。如果成功，集成的OCT和ExOCM成像平台将证明有助于 发展生物学、癌症研究和再生医学。此外，高吞吐量 样品的表征可以使用商业上可用的OCT系统进行，使ExOCM 易于传播和被更广泛的研究界采用。