title: Imaging MDCK cysts with a single (selective) plane illumination microscope.
abstract: 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.
authors.forename: Jim
authors.initials: J
authors.lastname: Swoger
authors.forename: Francesco
authors.initials: F
authors.lastname: Pampaloni
authors.forename: Ernst H K
authors.initials: EH
authors.lastname: Stelzer
mesh_terms: D000818:Animals; D004285:Dogs; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008029:Lighting / Q000379:methods*; D061985:Madin Darby Canine Kidney Cells; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*