Live imaging has proved powerful to solve many problems of cell biology, especially in the field of membrane trafficking. We have made several important observations such as demonstration of Golgi cisternal maturation (1), visualization of cargo transport from the ER exit site to the cis-Golgi by the hug-and-kiss action (2) and from an early zone to a late zone in a maturing Golgi cisterna (3) by pulse-chase imaging using the super-resolution confocal live imaging microscopy (SCLIM) we developed. Spatiotemporal resolution is critical in these studies, because the dynamics of vesicles often holds the clue to understand underlying mechanisms. We have recently built up a next-generation SCLIM, SCLIM2. SCLIM2 employs an ultrahigh-sensitivity, high-speed and multicolor detection system and enables us to perform 3-color 4D imaging at the resolution of 70-100 nm in space and 20 volumes/sec in time by single-photon counting and a new precise deconvolution algorithm we invented. I will present movies taken by SCLIM2, which show amazingly dynamic behaviors of Golgi cisternae, clathrin-coated buds and vesicles, as well as cargo, in yeast, plant and animal cells.
Refs: 1) Nature 441:1007-1010, 2006; 2) Nat. Commun. 5:3653, 2014; 3) J. Cell Biol. 218:1602-1618, 2019.