ABSTRACT
Super-resolution confocal live imaging microscopy (SCLIM) we developed provides high-speed, high-resolution, three- and four-dimensional, and multicolor simultaneous imaging. Using this technology, we are now able to observe the fine details of various dynamic events going on in living cells, such as membrane traffic and organelle dynamics. The retention using selective hooks (RUSH) system is a powerful tool to control synchronous release of natural cargo proteins of interest from the endoplasmic reticulum in mammalian cells. In this chapter, we describe a method for visualizing secretory cargo traffic within and around the Golgi apparatus in HeLa cells using SCLIM in combination with the RUSH assay.
Subject(s)
Endoplasmic Reticulum , Golgi Apparatus , Animals , Humans , HeLa Cells , Golgi Apparatus/metabolism , Protein Transport , Microscopy, Confocal/methods , Endoplasmic Reticulum/metabolism , MammalsABSTRACT
A recent key requirement in life sciences is the observation of biological processes in their natural in vivo context. However, imaging techniques that allow fast imaging with higher resolution in 3D thick specimens are still limited. Spinning disk confocal microscopy using a Yokogawa Confocal Scanner Unit, which offers high-speed multipoint confocal live imaging, has been found to have wide utility among cell biologists. A conventional Confocal Scanner Unit configuration, however, is not optimized for thick specimens, for which the background noise attributed to "pinhole cross-talk," which is unintended pinhole transmission of out-of-focus light, limits overall performance in focal discrimination and reduces confocal capability. Here, we improve spinning disk confocal microscopy by eliminating pinhole cross-talk. First, the amount of pinhole cross-talk is reduced by increasing the interpinhole distance. Second, the generation of out-of-focus light is prevented by two-photon excitation that achieves selective-plane illumination. We evaluate the effect of these modifications and test the applicability to the live imaging of green fluorescent protein-expressing model animals. As demonstrated by visualizing the fine details of the 3D cell shape and submicron-size cytoskeletal structures inside animals, these strategies dramatically improve higher-resolution intravital imaging.
Subject(s)
Imaging, Three-Dimensional/methods , Microscopy, Confocal/methods , Animals , Caenorhabditis elegans/cytology , Caenorhabditis elegans/embryology , Cell Survival , Drosophila melanogaster/cytology , Drosophila melanogaster/embryology , Embryo, Mammalian/cytology , Embryo, Nonmammalian/cytology , Green Fluorescent Proteins , Mice , Photons , Recombinant Fusion Proteins/metabolismABSTRACT
We have developed a high-speed confocal laser microscope. A microlens-array disk set in front of a pinhole-array disk improved optical efficiency more than ten times compared with that of conventional Nipkow confocal microscopy. This new microscope achieves a high-speed measurement of 1 frame/ms. We expect that it will be used for measuring biological and industrial active samples.
