This application requests a Zeiss LSM4l0 laser scanning confocal microscope and two Universal Imaging 486/66MHz workstations with Metamorph software. The facility will serve investigators from multiple departments throughout the Johns Hopkins Medical Institutions. Our 4 major users require confocal microscopy for l) work with dual-emission ion indicator dyes in living cells, and 2) high resolution colocalization of structures and multiple fluorescent labels in both fixed and living cells. Based on these needs, the LSM410 was selected to provide l) an inverted microscope platform for work with living cells, 2) multiple laser excitation including UV laser, 3) simultaneous collection of Nomarski transmitted light images and two confocal fluorescence signals, 4) automatic collection and storage of either XZ or XY image versus time, with on-line calculation of fluorescent emission ratios, and 5) a suitable instrument for a multi-user environment. All major users have tested the LSM41O to verify its suitability for their experiments. All post-acquisition data analysis will be performed on the requested workstations. The workstations will provide l) tools for extraction of morphological and intensity data from regions of interest within both large 3-D and 2-D image sets, 2) ability to analyze time course experiments with either XZ or XY images, 3) algorithms for co-localization of signals from different detectors and 4) an operating system which is both compatible with the LSM41O and familiar to users. Most workstation functions are included in Metamorph, but analysis of 3-D data sets and colocalization algorithms are provided by (free) PC-based software provide from an in-house faculty member who developed sophisticated image analysis programs for MRI applications. Combined with limited access to a Silicon Graphics workstation for volume rendering of 3-D data sets, these tools fulfill all our users' current need for image and data analysis. The confocal imaging and image analysis needs of our user can not be served by the single (5 year old) confocal at the Hopkins Medica Institutions, which is both overworked and inadequate. This new confocal facility will serve a scientific community which requires the advanced functions of the LSM41O and the sophisticated software available on the workstations.
