薄层荧光显微镜
显微镜
光学切片
光学
光漂白
荧光显微镜
显微镜
材料科学
亮场显微术
基底膜
共焦显微镜
荧光团
光学相干层析成像
荧光
生物
解剖
物理
作者
Jim Swoger,Francesco Pampaloni,Ernst H. K. Stelzer
出处
期刊:CSH Protocols
[Cold Spring Harbor Laboratory]
日期:2013-12-26
卷期号:2014 (1): pdb.prot080184-pdb.prot080184
被引量:7
标识
DOI:10.1101/pdb.prot080184
摘要
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). Madin-Darby canine kidney (MDCK) cysts grown in extracellular matrix (ECM) hydrogels provide a useful model system for studies of 3D cell biology. Here, we describe protocols for growing MDCK cysts within 3D type I collagen or reconstituted basement membrane (Matrigel) and for imaging these cysts by SPIM.
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