MULTIPHOTON CONFOCAL MICROSCOPE

Columbia University Health Sciences, New York NY

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Abstract

DESCRIPTION (provided by applicant): This is a request to replace a 2-photon confocal microscope that was purchased in 1999 with a new system incorporating several advances in multiphoton microscopy. The requested system consists of the Zeiss LSM 710 NLO confocal system and Coherent Chameleon Ultra II titanium-sapphire laser mounted on the Zeiss Axio Observer Z1 inverted microscope. The requested system offers both incremental improvements and funda- mental technological advances compared to our current system and other multiphoton systems. Among the significant advantages of the Zeiss system are 1) detectors with improved signal:noise ratio, 2) faster scanning, 3) more versatile laser line choice, 4) flexible emission collection, and 5) hardware and software that reduce service-related downtime. It will allow our users to carry out 2-photon imaging studies that cannot be performed with the existing microscope including 1) excitation from 900-1080 nm (e.g. YFP, RFP, DiI), 2) multicolor exci- tation, 3) detection of weaker signals, and 4) imaging thicker samples (>100-200 5m). Since it uses largely the same software and accessories as other microscopes in our core facility, it will provide an easy transition for users, while adding several important new capabilities. Finally, the system is cost-effective, as we can take ad- vantage of existing accessories (e.g. lenses, DIC optics, and environmental control for live-cell imaging). As a result, the cost of the requested instrument is significantly less than that of an equivalent new system. The Major Users have extensive expertise in optical imaging, and very sophisticated imaging needs. For example, our Major Users will use the requested instrument to carry out 4D imaging (time-lapse imaging combined with 3D reconstruction) of one or more fluorophores, of variable signal strength, in thick, living tissue samples over long time intervals (1-2 days). These studies are essential for visualization of cell differentiation, fate determination, migration, division, and degeneration during kidney development in organ culture, during tumor formation in skin, and in developing, mature and/or aging brain. Our Major Users are preeminent scientists who have made fundamental discoveries including the motor molecule dynein, stem cells in skin, and novel mechanisms for organelle movement. The Major Users include the first researchers to study kidney development in vitro, to isolate stem cells from skin, and to visualize spine motility in neurons using 2-photon microscopy. Not surprisingly, the research efforts of all Major Users are supported by NIH grants. Currently, no microscope at Columbia University has the capabilities of the requested instrument. Therefore, this equipment will enhance the user group's ability to complete projects underway, allow new projects to be initiated, and avoid duplication of effort and expense. The instrument will be incorporated into a productive core facility that has been directed by the PI since 1997, and receives significant institutional support. As a compo- nent of our Confocal and Specialized Microscopy Facility, the instrument will be accessible to Major Users and others throughout the University, promoting collaboration and exchange of ideas.