A diverse user-group of 12 investigators, within The Johns Hopkins University School of Arts &Sciences, Whiting School of Engineering, and the School of Medicine, request funds to purchase a Carl Zeiss Cell Observer SD. This system, based on the Carl Zeiss Axio Observer inverted research microscope, integrates a Yokogawa spinning disk confocal with: 1) 405nm/488nm/561nm/635nm lasers;2) two ultra-sensitive Evolve EMCCD cameras for simultaneous two-channel imaging;3) piezo-electric focusing for fast/precise Z-stack acquisition;and 4) a cage incubator for precise modulation of temperature, CO2, and humidity in extended live- imaging applications. All of these components are integrated, and conveniently accessed and precisely controlled through the Carl Zeiss AxioVision software. This new state-of-the-art workstation will be sited within the Hopkins'Integrated Imaging Center (IIC, www.jhu.edu/iic) on the Homewood campus;and constitute a significant upgrade to the live imaging capabilities for our Homewood/Hopkins-wide investigators whose needs have grown significantly over the previous decade. The IIC is a Homewood campus/Hopkins-wide microscopy resource, jointly supported by the Krieger School of Arts and Sciences and the Whiting School of Engineering (see Ball &Douglas letter, page 2 of the appendix);and utilized regularly by multiple schools and departments comprising >100 laboratories and >300 users. Our investigators, all well funded through the NIH, work on a host of diverse basic cell biology, biomolecular, and bioengineering related questions including, though not limited to: cell differentiation and gene regulation;Herpes virus assembly and maturation;mitochondrial fission;membrane organization and dynamics;microtubule-based motors;membrane trafficking;germ cell development and migration;sensory neuron development;molecular mechanisms of endocytosis;and circadian rhythm neuronal circuitry. In the requested configuration, the Cell Observer SD will afford our investigators the capability to precisely image multiple fluorophores simultaneously or sequentially at real-time acquisition speeds;and the ability to accurately modulate laser power to greatly minimize phototoxicity and photobleaching during extended time-lapse imaging sessions. Additionally, our investigators variously require the advanced live cell imaging techniques of FLIP, FRET, FRAP, and photoactivation of GFP, and 4D imaging. The new system will be incorporated into the IIC's existing, well established recharge system to ensure recovery of funds for supplies and maintenance;it will be made freely available to all interested users Hopkins- wide through our convenient web-scheduler;and it will be incorporated into the IIC's annual undergraduate/graduate course offerings. (www.jhu.edu/iic/academic.htm).
