Spinning disk confocal / FRAP microscope for quantitative live cell imaging

University Of California, San Francisco, San Francisco CA

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Abstract

DESCRIPTION (provided by applicant): We are requesting funds to purchase a state-of-the-art Spinning Disk Confocal Microscope System specifically designed for live cell imaging of mammalian cells and tissues. The instrument will be used by a core group of seven NIH-funded investigators and their laboratories to facilitate fourteen NIH- funded projects. The principle types of experiments for which this microscope system will be used include multidimensional imaging of fluorescently tagged proteins in living cells and tissues, analysis of protein activity by FRET, analysis of protein and organelle dynamics by FRAP or photoactivation, and photoablation or release of caged compounds in living cells or tissues. The system we are requesting includes a Yokogawa CSU-X1 spinning disk scanner on a Nikon Ti-E inverted microscope stand, which has a built-in focus feedback system allowing imaging for many hours at high resolution with no discernable focus drift. The microscope will also be equipped with an environmental chamber, a motorized stage, and CCD cameras for high resolution and high sensitivity imaging. Oil- and water- immersion objectives are requested for the optimal collection of data from live cells or tissues in a 2D or 3D environment at the highest achievable optical resolution. Solid-state laser lines are requested at 440, 488, 514, and 561 nm to permit imaging of all commonly used fluorescent protein variants. In addition, this instrument will be equipped with a pulsed dye laser-based photobleaching / photoablation module extending its usefulness beyond simple observation of live specimens. Currently, no spinning disk microscope system exists at UCSF that has FRAP or photoactivation capabilities, which severely limits the types of experiments that can be performed. It is expected that the major users will utilize the instrument for ~80% time. However, because the instrument will be housed in and managed through the UCSF Parnassus Biological Imaging Development Center, training and use will be available to additional NIH-funded investigators within the UCSF community.