Development of highspeed and super-resolution microscopy

Abstract

Live cell imaging is a powerful tool to understand the molecular mechanisms of a wide range of cellular processes. However, it is difficult to detect very small objects such as transport vesicles (< 100 nm in diameter) by light microscopy because of the diffraction limit of light. To overcome this problem, several superresolution technologies such as STED, SIM, and STORM/PALM has been developed so far, but the time resolution of these methods is not sufficient to track the small vesicles undergoing fast movement inside the cell. In the present study, we developed a highspeed, superresolution, and multicolor optical microscope, which we call SCLIM2 (the second generation superresolution confocal live cell imaging microscopy). SCLIM2 consists of an inverted microscope equipped with piezo actuator for objective lens, a spinningdisk confocal scanner, an emission splitter unit, cooled image intensifiers, and ultrafast CMOS cameras (1000 frames/s). Using this system, we acquired fluorescence images of specimens at a single photon precision. We also developed a mathematical method for 4D (xyz and time) image reconstruction and novel deconvolution algorism based on the probability calculation. The resulting lateral resolution was less than 71 nm. We then applied SCLIM2 to observe a variety of membrane traffic events in live cells. We succeeded in visualizing a large number of small vesicles (< 100 nm in diameter) were moving around the Golgi apparatus.