Wide field-of-view Spinning Disk Confocal with Photoactivation and Photobleaching

Thorn, Kurt

Abstract

We are requesting funds to purchase a wide field-of-view high speed spinning disk confocal microscope. This confocal will be installed on an existing microscope stand with photoactivation and photobleaching capabilities. This microscope will be used for live cell imaging by a group of 8 investigators to carry out a total of 17 NIH-funded research projects. It will be housed in and managed by the Nikon Imaging Center (NIC), a highly successful core facility at UCSF. The NIC houses an existing spinning disk confocal microscope, used by many of the major investigators on this proposal. This microscope is extremely heavily used (315 hours/month on average) and these users are unable to get sufficient time on this microscope to carry out their research. In addition to providing improved access, the proposed system provides several benefits over our current microscope: it has a ~7-fold larger field-of-view, enabling imaging of larger samples and collecting much more data and it can operate at higher speeds, allowing rapid multicolor and 3 dimensional imaging. Finally, this system is equipped with a photoactivation and photobleaching system, allowing direct probing of protein dynamics in live cells. UCSF has awarded two grants, totaling $290 000, to purchase the microscope stand that will serve as the base for this system. This stand includes a fully motorized Nikon Ti-E microscope with hardware autofocus, a motorized XY stage with a piezoelectric Z drive, a stage-top incubator, and a Rapp Optoelectronic photobleaching / photoactivation system with 405 nm and 473 nm lasers. Here we are requesting funds to purchase a spinning disk confocal to add to this microscope. The requested confocal will use the Yokogawa CSU-W1 scanhead, and will be capable of imaging a 130 x 130 m field of view at 100x, using a 4 megapixel Andor sCMOS camera. We are requesting a high power four channel laser launch (405 / 488 / 561 / 647 nm) for four-color imaging of fluorescent proteins and small molecule dyes. This hardware will be integrated with the microscope stand to allow acquisition of multicolor Z-stacks at speeds of up to 100 fps. The combined system is capable of bleaching or photoactivation simultaneously while imaging and is the first photoactivation or photobleaching system in a core facility at the Mission Bay campus of UCSF. We expect that the major users on this application will use ~85% of the time available on this instrument. However, since it will be housed in and managed by the NIC, additional time on the system will be available to other researchers, and we expect that this system will have a major impact on UCSF research more broadly.