Axial Slice Light Sheet Microscopy

描述（申请人提供）：生物细胞和组织的三维光学成像在生物医学研究和医学诊断中越来越重要。虽然横向（即平行于样品表面）的图像通常可以平行（例如，使用薄片显微镜）或高速（例如，使用高速扫描显微镜）获得，但目前没有一种成像方式可以平行成像轴向切片（即垂直于样品表面），这限制了我们可视化不同深度发生的快速生物过程的能力。在本研究中，我们的目标是通过开发一种能够平行成像样品轴向切片的新技术来克服这一限制。关键概念是使用沿轴向排列的45°倾斜高纵横比微镜阵列，该阵列可以利用不同波前从不同深度位置发射的图像信号进行多路分解。每个微镜的尺寸与衍射极限光束宽度相当。因此，它的行为有效地作为一个共聚焦针孔或狭缝，提供光学切片能力。在我们初步研究的基础上，在提出的方案中，我们将首先设计提出的高纵横比微镜阵列，并通过微成型制造它。然后，我们将结合高纵横比微镜阵列器件开发所提出的轴向切片光片成像系统。将对原型系统进行表征和优化。高分辨率轴向切片光片成像荧光微球和三维球形肿瘤细胞培养复杂系统将被证明。

DESCRIPTION (provided by applicant): Three-dimensional optical imaging of biological cells and tissues has become increasingly critical for biomedical studies and medical diagnosis. Although images in the lateral direction (i.e., parallel to the sample surface) can be routinely acquires in parallel (e.g., using light-sheet microscopy) or at high speed (e.g., using high-speed scanning microscopy), no current imaging modality can image an axial slice (i.e., perpendicular to the sample surface) in parallel, limiting our ability to visualize fast biological processes happening across different depths. In this proposed study, we aim to overcome this limitation by developing a new technology capable of imaging axial slices of a sample in parallel. The key concept is the proposed use of an array of 45¿- tilted high-aspect-ratio micro mirrors arranged along the axial direction, which can demultiplex image signals emitted from different depth positions by exploiting their different wavefronts. Each micro mirror has a dimension comparable to the diffraction limited beam width. Consequently it behaves effectively as a confocal pinhole or slit, providing optical sectioning capability. Building upon our preliminary study, in the proposed program we will first design the proposed high-aspect-ratio micro mirror array and fabricate it by using micromolding. We will then develop the proposed axial slice light-sheet imaging system by incorporating the high-aspect-ratio micro mirror array device. The prototype system will be characterized and optimized. High- resolution axial slice light sheet imaging of fluorescent microspheres and 3D spheroid tumor cell culture complex systems will be demonstrated.