A domed window chamber for multi-modality optical imaging

Current dorsal skin flap window chambers with flat glass windows are compatible with optical coherence tomography (OCT) and multiphoton microscopy (MPM) imaging. However, light sheet fluorescence microscopy (LSFM) performs best with a cylindrical or spherical sample located between its two 90° objectives and when all sample materials have the same index of refraction (<i>n</i>). A modified window chamber with a domed viewing window made from fluorinated ethylene propylene (FEP), with n similar to water and tissue, was designed. <i>In vitro</i> imaging of collagen gels and microsphere phantoms with and without the dome showed small decreases in signal strength and image resolution due to the dome. Using a custom mouse platform for stabilization and anesthesia support, <i>in vivo</i> multimodality imaging with OCT, MPM, and LSFI was demonstrated. Experimental techniques for live tumor imaging have traditionally used flat glass skin window chambers. These windows enable tumor assessment in the natural environment using light-based imaging modalities including optical coherence tomography (OCT) and multiphoton microscopy (MPM), however, they are not compatible with the geometry of light sheet fluorescence microscopy (LSFM). Multi-instrument imaging may improve our ability to characterize and understand tumors. We developed an optically clear plastic dome-shaped window to accommodate LSFM imaging in addition to OCT and MPM since the raised dome fits between the two objectives of the light sheet microscope. The dome-shaped window chamber was tested with microsphere targets to understand the impact of the dome on the image quality of OCT, MPM, and LSFM. It was also implanted onto the skin of live mice with tumors and imaging was demonstrated. The window chamber and a custom-designed mouse imaging platform were compatible with OCT, MPM, and LSFM imaging of live mice. The dome-shaped plastic window caused minor (less than 10%) degradation of the resolution and signal strength in all three imaging modalities. Overall, our experiments confirmed that assessment of live tumors using LSFM imaging, in addition to OCT and MPM imaging, can be made possible by using a dome-shaped window. Window chambers enable the visualization of interior body structure and function in mouse research models. A modified dorsal skin flap window chamber was designed and built, containing an optically clear dome made from fluorinated ethylene propylene (FEP). This window chamber was tested for compatibility and image quality with three optical imaging modalities: optical coherence tomography, multiphoton microscopy, and light sheet fluorescence microscopy. The index of refraction of FEP (similar to water) and the domed shape enabled quality imaging with all three modalities. <i>In vivo</i> imaging was demonstrated using the window chamber installed on a mouse, and facilitated by a custom <i>in vivo</i> imaging fixture, including a window chamber holder and a stable imaging base. A custom domed dorsal skin flap window chamber and custom <i>in vivo</i> imaging fixture were designed and fabricated. Imaging was successfully performed with optical coherence tomography (OCT), multi-photon microscopy (MPM), and light sheet fluorescence microscopy (LSFM) imaging modalities, both <i>in vitro</i> with phantoms, and <i>in vivo</i> with a wild-type mouse.当前采用平面玻璃窗的背侧皮瓣开窗腔室可兼容光学相干断层扫描（optical coherence tomography, OCT）与多光子显微镜（multiphoton microscopy, MPM）成像。然而，光片荧光显微镜（light sheet fluorescence microscopy, LSFM）的最优成像条件为：样品呈圆柱或球形，且置于其两台90°物镜之间，同时所有样品材料的折射率（n）保持一致。本研究设计了一款改良型开窗腔室，其采用氟化乙烯丙烯（fluorinated ethylene propylene, FEP）制成的穹顶式观察窗口，该材料的折射率与水及生物组织相近。通过对有无穹顶的胶原凝胶与微球体模开展体外成像，发现穹顶会导致信号强度与图像分辨率出现小幅下降。借助定制化小鼠固定与麻醉支撑平台，本研究成功实现了OCT、MPM与LSFM的活体多模态成像。传统活体肿瘤成像实验技术多采用平面玻璃皮肤开窗腔室，此类窗口可借助光学相干断层扫描（OCT）、多光子显微镜（MPM）等光学成像模态在自然生理环境中实现肿瘤评估，但无法适配光片荧光显微镜（LSFM）的成像几何要求。多仪器联合成像可提升我们对肿瘤的表征与认知能力。鉴于穹顶结构可嵌入光片显微镜的两台物镜之间，本研究开发了一款光学透明的塑料穹顶式窗口，使其除支持OCT与MPM成像外，亦可适配LSFM成像。本研究通过微球靶标对该穹顶式开窗腔室开展测试，以明确穹顶对OCT、MPM及LSFM成像质量的影响。此外，该腔室还被植入荷瘤活小鼠的皮肤部位，并成功实现成像。该开窗腔室与定制化小鼠成像平台可兼容活体小鼠的OCT、MPM与LSFM成像。该塑料穹顶式窗口会导致三种成像模态的分辨率与信号强度出现小幅衰减（衰减幅度小于10%）。综上，本研究实验证实：通过采用穹顶式窗口，即可实现LSFM联合OCT与MPM对活体肿瘤的评估。开窗腔室可实现小鼠研究模型体内组织结构与功能的可视化观测。本研究设计并构建了一款改良型背侧皮瓣开窗腔室，其采用氟化乙烯丙烯（FEP）制成的光学透明穹顶。本研究针对该腔室与三种光学成像模态的兼容性及成像质量开展测试，这三种模态分别为光学相干断层扫描、多光子显微镜与光片荧光显微镜。FEP的折射率与水相近，结合穹顶式结构，使得该腔室可兼容上述三种成像模态并获得优质成像效果。本研究通过定制化活体成像固定装置（含窗口腔室固定架与稳定成像基座），将该开窗腔室安装于小鼠体表，成功实现活体成像。本研究设计并制备了定制化穹顶式背侧皮瓣开窗腔室与定制化活体成像固定装置。本研究分别通过体模与野生型活小鼠，成功实现了光学相干断层扫描（OCT）、多光子显微镜（MPM）与光片荧光显微镜（LSFM）三种成像模态的成像。