A Confocal Laser Scanning Microscope for Neuroscience Imaging Center

Armstrong, William

Abstract

The proposal is to purchase a modern confocal laser confocal scanning microscope (Zeiss LSM 710) with wide spectral scanning capability, high resolution, and high sensitivity for a Neuroscience Imaging Center at the University of Tennessee Health Science Center. The LSM will be mounted on a Zeiss Axio Observer inverted microscope, providing great flexibility in the type of material examined. The microscope stage will have several adaptors such that live neurons may be imaged in a warmed, oxygenated, humidified environment in a variety of incubation wells, or by switching adapters, fixed brain slices may be imaged with specific structures and/or molecules labeled with fluorescent probes. The LSM comes equipped with six laser lines, and three photomultipliers (PMTs) capable of scanning 34 channels, and is much more sensitive than previous LSMs. This sensitivity means that less laser power, which is damaging to live cells and which bleaches the probes used in these studies, is required. These fluorescent probes can span a variety of emitted wavelengths, and up to 10 probes may be simultaneously viewed and characterized. The spectral scanning sensitivity is such that probes with fluorescent emission profiles 10 nm or even less may be separated, graphically marked, and analyzed. Thus, neuroscientists will have great flexibility to determine the precise location, and biochemical nature, of neurons, their extensive processes, and their synaptic connections. The spatial resolution of this LSM (6144 x 6144) combined with Zeiss objectives and confocal optics, means that structures well below the limit of conventional light microscopes (~0.10 5m) may be viewed, measured and counted. Neuroscientists here will be studying a broad array of problems, from how neurons develop appropriate connections, to understanding which ion channels sculpt their electrical behavior necessary for communication. This information is critical to understanding how neurodegenerative or neuropathic diseases such Parkinson's, Huntington's, and dystonia arise and progress - information critical to understanding how we might cure these conditions. Brain proteins abnormally expressed in a variety of genetic conditions, such as those related to some forms of autism, also will be examined with this microscope. Live imaging capability means that drugs may be tested for their efficacy in fighting brain tumor cells, with real-time results. The basic structures of many brain areas, such as those serving olfaction, nervous system control of pituitary hormones, sensory-motor integration, coordination of movement and cognition will be examined, as will normal brain development, and the deleterious effects of ethanol.