Expansion Optical Coherence Microscopy (ExOCM)

点击翻译按钮获取中文摘要

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.