Dual Confocal Microscopic Scanner

Rush University Medical Center, Chicago IL

Investigators

Linked publications & trials

Abstract

[unreadable] DESCRIPTION (provided by applicant): Confocal systems include a microscope, a group of lasers, and a scanner, which guides the excitation light into the microscope and the emitted light into one or more photosensors. In the instrument that we seek, Zeiss LSM 5-Meta/LIVE combination (or DUO), two scanners, each with a set of lasers, converge into one microscope. The main purpose of the 2nd scanner (operator) is to provide additional irradiation of the sample, while independently imaging fluorescence via the first scanner. The operator permits a number of operations on the sample, all related to photoconversion. There are no instruments competing with the one requested, which is the only dual scanner available. Major users are experienced imagers from three departments and divisions of Rush University plus the head of Loyola University's Imaging Core, who will apply the salient features of the instrument for the purposes of (i) calcium monitoring inside cytosol, mitochondria and storage organelles of skeletal and cardiac muscle and brain vascular endothelia, (ii) proton concentration and evolution in phagosomes and submembrane domains, (iii) membrane curvature and lipid rafts on living cells and cell fractions by FRET between raft-preferred or raft-excluded molecules, (iv) monitoring of unidirectional flux through individual channels in bilayers and (v) monitoring of cAMP in endothelial cells. This monitoring will be simultaneous with operator-induced photorelease of Ca2+, protons, or IP3. In various cases monitoring will use the new modality of SEER (developed by the PI) to maximize probe sensitivity. While some of these operations can be done with conventional confocal microscopes, most require the ability to simultaneously and independently define the operator light and the imaging parameters of excitation and emission. Projects will also benefit from the unique flexibility of excitation and emission wavelengths, especially valuable for state of the art biosensors already implemented, and the extreme imaging speed, useful for following rapidly evolving microdomains as well as mobile organelles and rafts. The projects are directly or indirectly relevant to a number of disease entities, including ALS (i), HIV infectivity (iv), heart failure and alterations of rhythm (i, ii), inflammatory and immune response (iii, v). The extraordinary level of institutional support bestowed on this proposal attests to its perceived importance for basic and translational research at Rush University. [unreadable] [unreadable] [unreadable]