Live Cell Imaging Confocal Microscope with Spectral Detector and Resonant Scanner

Lagunoff, David

Abstract

This is a proposal to implement an advanced live cell, spectral imaging, resonant scanning laser confocal microscope within the Imaging Core Facility at New Jersey Medical School. The microscope will be used to enhance the capabilities of researchers to analyze pathogenesis at the cellular level in order to develop means of prevention and of blocking disease progression. Diseases under study by major users of the confocal include HIV-AIDS (1), leukemia (2), tuberculosis( 3,4), perinatal hypoxic brain injury(5), heart failure(5,6,7,8,9), alcoholic liver disease(10,11) and toxoplasmosis(12,13). Minor users will utilize the microscope for their studies of an important pediatric pathogen, K. kingae, botulinum toxin, the reaction to electrode implantation in the CNS, gastrointestinal radiation injury, and osteoporosis. The Nikon A1Rsi confocal microscope we are requesting has the ability to provide detailed dynamic as well as static images of fluorescent molecules, the coordinated movement of such molecules, the interaction of appropriately labeled intracellular molecules with one another and with cell organelles, and to associate molecular changes with deviant behavior of cells. Several of the projects of major users will employ fluorescent fusion proteins in live cells;other investigators will utilize fluorescent sensors to monitor changes in intracellular pH, Ca++, cAMP or redox potential in living cells, and yet others will employ fluorescent antibodies or fluorescent chemical probes to specifically locate surface, cytoplasmic, organelle and nuclear molecules in fixed cells. The requested microscope will enhance localization of fluorescent molecules in 3-dimensions and add the ability to track fluorescent molecules over time in live cells and to identify specific fluorescent molecules in multi-labeled samples utilizing multiple lasers and high resolution fluorescent emission spectra. Software is available for experiments requiring measures of molecular association (FRET) and for kinetic collection of fluorescent intensities of probes with the resonant scanner following photoactivation using the galvanometer scanner. The combination of a motor driven stage and stitching of images allows monitoring of cells over greater areas than possible with single frame collection of images with high N.A. objectives. The currently available LSM510 with 2 lasers and 2 detector channels is not able to provide sufficient capability to meet the needs of neither users identified in this application nor others served by the core facility. Because of the configuration and vintage of the LSM510, according to Zeiss Microimaging, it is not possible to upgrade either the hardware or the software, nor is a service contract available, subjecting the instrument to extended downtime and possible total loss.