Development of Multi-Functional Microscopy (MFM) for Cancer and AIDS Research
EMSL Project ID
6898
Abstract
The goal of this project is to advance technology for studying complex molecular mechanisms at the cell and tissue levels. Such mechanisms underlie the processes of carcinogenesis, HIV infection and other diseases. They are governed by the spatial-temporal distributions and interactions of many molecules in cells, and cell-to-cell interactions. The technology is a combination of molecule-specific labeling, optical microscopy, quantitative image analysis and mathematical modeling. However, the power of the technology is limited by the low number of molecular species that can be investigated simultaneously in intact cells and tissues. This project will accomplish its goal through a collaboration between the NCI and PNNL to develop multi-functional microscopy (MFM) and related methods for simultaneously imaging multiple molecular species (with and without fluorescence labeling), thereby increasing our understanding of the complex molecular mechanisms of cells. The principal technical development is coherent anti-Stokes Raman spectroscopy (CARS) microscopy for detecting endogeneous and deuterated functional groups in molecules. This technology will be used in combination with fluorescence microscopy and second harmonic generation (SHG) microscopy for detecting unlabeled, organized molecular complexes in cells. The key element of CARS and SHG is non-linear, multi-photon methods that do not require fluorescence labeling for imaging of specific cellular constituents. The individual data channels mark different chemical and physical properties of cellular constituents and are expected to provide distinct signatures of the molecular and physical state of the cells. We will demonstrate the utility of MFM in feasibility studies in the areas of Alzheimer?s disease and cancer. The latter project will study how arachidonic acid metabolism contributes to the transformation response of JB6 mouse epidermal cells.We request one NCI postdoctoral fellow, who will primarily work at PNNL to undertake to assess the feasibility of CARS microscopy to answer questions about the cellular mechanisms of cancer and Alzheimer?s disease. We estimate the cost will be $70,000 per year for two years.
We also expect that MFM will be used for the intracellular localization of drugs that cannot be fluorescence labeled. Therefore at the end of this document is a proposal from Dr. Chris Michejda outlining two experiments where CARS microscopy would overcome these limitations. If resources are available, we would undertake these studies.
Project Details
Project type
Exploratory Research
Start Date
2004-01-21
End Date
2006-01-23
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Amonette JE, O Qafoku, TW Wietsma, PM Jeffers, CK Russell, and MJ Truex. 2010. Abiotic Degradation Rates for Carbon Tetrachloride and Chloroform: Progress in FY2009. PNNL-19142, Pacific Northwest National Laboratory, Richland, WA. 42 pp.
Camilloni C, AB Sahakyan, M Holliday, NG Isern, F Zhang, EZ Eisenmesser, and M Vendruscolo. 2014. "Cyclophilin A catalyzes proline isomerization by an electrostatic handle mechanism." Proceedings of the National Academy of Sciences of the United States of America 111(25):, doi:10.1073/pnas.1404220111
Holliday M, F Zhang, NG Isern, GS Armstrong, and EZ Eisenmesser. 2014. "1H, 13C, and 15N backbone and side chain resonance assignments of thermophilic Geobacillus kaustophilus cyclophilin-A." Biomolecular NMR Assignments 8(1):23-27. doi:10.1007/s12104-012-9445-3
Rodriguez L, SJ Lockett, and GR Holtom. 2006. "Coherent Anti-Stokes Raman Scattering Microscopy: A Biological Review." Cytometry. Part A 69(8):779-791.