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 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，我们将获得超分辨率，分子特异性图像的扩展组织 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观察，证明了基于固有散射对比度的ExOCM的可行性。进一步 能够实现分子特异性成像，造影剂，如金纳米颗粒（GNP），分子染料， 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可以高速成像超厚样品。这种方法还允许 三维（3D）分子分布与从显微镜获得的活组织微观结构的配准 基于我们集团在开发OCT和 OCM技术和3D类器官模型的经验，我们建议开发，优化和验证 ExOCM技术，并展示了ExOCM用于生物医学研究的独特属性。两个具体 该计划的目标是：目标1。开发和优化ExOCM技术，以实现多色、超 扩展的类器官的分辨率成像。我们将优化超高分辨率OCM系统，以实现~1.5 µ m各向同性图像分辨率。抗体缀合的GNP将用于提供特异性靶向。 proteins.使用我们优化的OCM系统和~4.5X组织扩张方案，我们预计将获得~300 nm分辨率的轴向和横向尺寸。将获得多色和多路复用成像 使用光谱OCT分析从具有不同吸收和散射的GNP分离信号 数据区.目标2.为了验证ExOCM与双光子显微镜，并证明的可行性， 组合OCT和ExOCM成像方法，以表征3D结构和分子轮廓以及生长 相同样品的动力学。我们将使用OCT获取活体类器官的纵向无标记图像 在三周的时间里。在预定的时间点，样品将扩增用于ExOCM和两个- 光子成像，其中将从用于配准的3D图像堆栈中识别组织结构标志。 在扩展过程中收集的ExOCM成像数据将提供有关3D的第一手信息 膨胀均匀性互补的3D结构和分子轮廓以及来自相同的生长动力学 将获得类器官，证明OCT和ExOCM成像组合的独特优势 approach.如果成功，集成的OCT和ExOCM成像平台将证明有助于 发育生物学、癌症研究和再生医学。此外，高通量 可以使用市售的OCT系统进行样本的表征，使得ExOCM 易于传播和采用更广泛的研究社区。