Swoger Jim, Pampaloni Francesco, Stelzer Ernst H K
Cold Spring Harb Protoc. 2014 Jan 1;2014(1):106-13. doi: 10.1101/pdb.prot080176.
In modern biology, most optical imaging technologies are applied to two-dimensional cell culture systems. However, investigation of physiological context requires specimens that display the complex three-dimensional (3D) relationship of cells that occurs in tissue sections and in naturally developing organisms. The imaging of highly scattering multicellular specimens presents a number of challenges, including limited optical penetration depth, phototoxicity, and fluorophore bleaching. Light-sheet-based fluorescence microscopy (LSFM) overcomes many drawbacks of conventional fluorescence microscopy by using an orthogonal/azimuthal fluorescence arrangement with independent sets of lenses for illumination and detection. The specimen is illuminated from the side with a thin light sheet that overlaps with the focal plane of a wide-field fluorescence microscope. Optical sectioning and minimal phototoxic damage or photobleaching outside a small volume close to the focal plane are intrinsic properties of LSFM. The principles of LSFM are implemented in the single (or selective) plane illumination microscope (SPIM). Cellular spheroids are spherical aggregations of hundreds to thousands of cells and they provide a useful model system for studies of 3D cell biology. Here we describe a protocol for imaging cellular spheroids by SPIM.
在现代生物学中，大多数光学成像技术应用于二维细胞培养系统。然而，对生理环境的研究需要能够展示组织切片和自然发育生物体中细胞复杂三维（3D）关系的标本。对高度散射的多细胞标本进行成像存在诸多挑战，包括光学穿透深度有限、光毒性和荧光团漂白。基于光片的荧光显微镜（LSFM）通过使用正交/方位荧光配置，配备独立的照明和检测透镜组，克服了传统荧光显微镜的许多缺点。标本由与宽场荧光显微镜焦平面重叠的薄光片从侧面照明。光学切片以及焦平面附近小体积区域外的最小光毒性损伤或光漂白是LSFM的固有特性。LSFM的原理在单（或选择性）平面照明显微镜（SPIM）中得以实现。细胞球状体是由数百到数千个细胞组成的球形聚集体，它们为三维细胞生物学研究提供了一个有用的模型系统。在此，我们描述一种通过SPIM对细胞球状体进行成像的方案。
