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.