TY  - JOUR
TI  - Live-cell Imaging by Super-resolution Confocal Live Imaging Microscopy (SCLIM): Simultaneous Three-color and Four-dimensional Live Cell Imaging with High Space and Time Resolution
AU  - Kurokawa, Kazuo 
AU  - Nakano, Akihiko 
VL  - 10
IS  - 17
PY  - 2020
DA  - 2020/09/05
SP  - e3732
C1  - Bio-protocol 2020;10:e3732
DO  - 10.21769/BioProtoc.3732
UR  - https://doi.org/10.21769/BioProtoc.3732
AB  - Many questions in cell biology can be solved by state-of-the-art technology of live cell imaging. One good example is the mechanism of membrane traffic, in which small membrane carriers are rapidly moving around in the cytoplasm to deliver cargo proteins between organelles. For directly visualizing the events in membrane trafficking system, researchers have long awaited the technology that enables simultaneous multi-color and four-dimensional observation at high space and time resolution. Super-resolution microscopy methods, for example STED, PALM/STORM, and SIM, provide greater spatial resolution, however, these methods are not enough in temporal resolution. The super-resolution confocal live imaging microscopy (SCLIM) that we developed has now achieved the performance required. By using SCLIM, we have conducted high spatiotemporal visualization of secretory cargo together with early and late Golgi resident proteins tagged with three different fluorescence proteins. We have demonstrated that secretory cargo is indeed delivered within the Golgi by cisternal maturation. In addition, we have visualized details of secretory cargo trafficking in the Golgi, including formation of zones within a maturing cisterna, in which Golgi resident proteins are segregated, and movement of cargo between these zones. This protocol can be used for simultaneous three-color and four-dimensional observation of various phenomena in living cells, from yeast to higher plants and animals, at high spatiotemporal resolution.
KW  - Live cell imaging
KW  - Simultaneous 3-color and 4-dimensional observation
KW  - Super-resolution and high-speed observation
KW  - Secretory cargo
KW  - Golgi
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False