Superfund Research Program
Image Analysis and Bioassays Core
Project Leader: Robert C. Burghardt
Grant Number: P42ES004917
Funding Period: 2000-2008
The Image Analysis Core continues to work closely with SBRP investigators to provide routine and advanced analytical microscopy and image analysis support services. Improvement in analytical microscopy instrumentation and services including specialized training programs is an ongoing priority. Based upon a needs assessment carried out during the previous year, a shared instrumentation grant proposal to NIH, National Center for Research Resources, was submitted for a confocal/multiphoton microscope with accessories designed to support the needs of multiple SBRP investigators. Funding was awarded in the amount of $499,137 along with institutional matching support of $272,000 to purchase a Zeiss LSM 510 META NLO Confocal/multiphoton microscope equipped with a Coherent Chameleon Ultra hands-free Ti:Sapphire laser and Verdi pump laser, multi-line argon laser, and dual HeNe lasers with 3 photomultiplier tubes interfaced with an Axiovert 200 M inverted microscope. Accessories include the META module which provides analysis of spectral signatures and separation of overlapping spectral emissions and a comprehensive software package that includes, multiple time series, physiology, FRET, FRAP analyses. The configuration of this system was specifically designed to address identified needs of SBRP investigators. New opportunities provided by this technology includes the ability to scan emission spectra of PAH exposed tissues to identify spatial distribution of metabolites with a computer controlled non-invasive, non destructive femtosecond pulsed laser system. Other applications include improved software analysis of protein-protein interactions with FRET assays and a variety of cell physiology software for kinetic analysis of fluorescence endpoints of cellular homeostasis. During the current funding period, the "Endocrine Disruptors: Mechanistic Studies" and "Genotoxicity of Complex Mixtures" projects made extensive use of several different vital imaging instruments (live cell and digital imaging workstations, confocal and multiphoton microscopes) and applications for routine bioassays of cellular function in toxicant-exposed cell types including: the FRAP approach to quantify effects of PAH mixtures on intercellular communication (a tumor promoter assay) and assessment of cytochrome P450 enzyme induction (EROD assay); single-cell and bulk analysis of cytochrome P450 enzyme induction; reactive oxygen species production, apoptosis assays, and analysis of intracellular Ca2+ homeostasis and Ca2+ waves and oscillations. The "Endocrine Disruptors: Mechanistic Studies" project also continued utilization of immunocytochemistry as well as direct analysis of estrogen receptor (ER), aryl hydrocarbon (AhR), Sp1-Sp4 and coactivator protein-protein interactions using FRET assays. Other analyses involved the evaluation of the efficacy of specific siRNA constructs on the expression and function of these proteins. The "Sensitive Genotypes to Arsenic as a Model Environmental Teratogen" project continued further analysis of ultrastructural changes and apoptosis in neural tubes of fetal mice exposed to arsenic.