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Three-Dimensional Visualization and Volumetric Analysis of Cancer Therapeutic Agents and Cell Interactions Using Raman Imaging, 10-9110

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Principal Investigator
Jian Ling

Inclusive Dates: 01/01/99 - Current

Background - Every day approximately 1,500 Americans die from cancer, and these deaths are often prolonged and painful. Cancer therapeutics is a severe challenge to the healthcare industry. Recently, the implementations of rational drug design, combinatorial chemistry techniques, and high throughput screening have led to large numbers of new, potential anticancer agents. However, the mechanisms by which these agents kill tumor cells are unknown.

Approach - The objective of this project is to develop techniques that would enable the use of Raman microscopic imaging to study the actions of anticancer drugs at the cellular level. Specifically, a Raman imaging technique will be developed to study the uptake (amount of the drug taken into the cell), subcellular localization (specific location at which the drug acts), and characteristics of local binding (biochemical interaction) of anticancer drugs within the cells. Anticancer drugs with known cell interactions will be used in this study to evaluate the feasibility of the Raman imaging technique. Raman image is a chemical image with the potential to display the spatial distribution of a substance that is invisible in conventional optical images. This unique characteristic is because the Raman spectra intrinsically depends upon the structure (vibrational states and chemical bonds) of the molecules that make up a substance. In this study, two Raman images, tuned at the unique Raman peaks of the anticancer drug, will be obtained from a cancer cell before and after the drug treatment. The differences between the two images will show the two-dimensional distribution of the drug within the cell. A stack of such images representing different cellular cross sections will be obtained from the Raman microscope for three-dimensional (3-D) construction. The reconstructed 3-D Raman region indicates the 3-D drug distribution within the cell. From the drug distribution region, the volume and concentration of the drug can be estimated. A 3-D visualization program will be developed to display the locations of the drug within the cell. Knowing the drug’s distribution is an important factor in evaluating its efficacy.

Accomplishments - The Raman microscopic system was established for imaging the biological samples. Polystyrene microspheres were used to calibrate the microscopic system. Image-processing algorithms such as Raman image restoration and enhancement were developed. Raman spectra of both anticancer drugs and cellular backgrounds were measured. From the spectra, the fingerprint Raman band was identified. At the drug's unique Raman band, Raman images of a cell were taken. The illustration shows the Raman images of a breast cancer cell before and after drug treatment. The high-intensity spots in the Raman image after the drug treatment suggest the distribution of the drug.

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A MDA435 breast cancer cell is shown before and after drug treatment. The top-left white-light image shows the cell in control. Its corresponding Raman image at 1002 cm-1 (within the red-box) is illustrated in the bottom-left image, while the top-right white-light image shows the cell after drug treatment. Its corresponding Raman image is shown in the bottom-right image.

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