Swoger Jim, Pampaloni Francesco, Stelzer Ernst H K
Cold Spring Harb Protoc. 2014 Jan 1;2014(1):1-8. doi: 10.1101/pdb.top080168.
In modern biology, most optical imaging technologies are applied to two-dimensional cell culture systems; that is, they are used in a cellular context that is defined by hard and flat surfaces. However, a physiological context is not found in single cells cultivated on coverslips. It requires the complex three-dimensional (3D) relationship of cells cultivated in extracellular matrix (ECM) gels, tissue sections, or in naturally developing organisms. In fact, the number of applications of 3D cell cultures in basic research as well as in drug discovery and toxicity testing has been increasing over the past few years. Unfortunately, the imaging of highly scattering multicellular specimens is still challenging. The main issues are the limited optical penetration depth, the phototoxicity, and the 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 basic idea is to illuminate the specimen 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. We discuss the basic principles of LSFM and methods for the preparation, embedding, and imaging of 3D specimens used in the life sciences in an implementation of LSFM known as the single (or selective) plane illumination microscope (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；也就是说，它们用于由坚硬且平坦表面定义的细胞环境中。然而，在盖玻片上培养的单细胞中找不到生理环境。它需要细胞在细胞外基质（ECM）凝胶、组织切片或自然发育的生物体中培养时形成的复杂三维（3D）关系。事实上，在过去几年中，3D细胞培养在基础研究以及药物发现和毒性测试中的应用数量一直在增加。不幸的是，对高度散射的多细胞标本进行成像仍然具有挑战性。主要问题是光学穿透深度有限、光毒性和荧光团漂白。基于光片的荧光显微镜（LSFM）通过使用具有独立照明和检测透镜组的正交/方位角荧光装置，克服了传统荧光显微镜的许多缺点。其基本思想是用一个薄光片从侧面照射标本，该光片与宽场荧光显微镜的焦平面重叠。光学切片以及在靠近焦平面的小体积区域外最小的光毒性损伤或光漂白是LSFM的固有特性。我们将在一种称为单（或选择性）平面照明显微镜（SPIM）的LSFM实现方式中，讨论LSFM的基本原理以及生命科学中使用的3D标本的制备、包埋和成像方法。
