Date of Award
Doctor of Philosophy
Gerald J. Small
The high resolution spectroscopic technique of nonphotochemical hole burning (NPHB) was applied to the study of in vitro cellular systems. Suspensions of two well-characterized cell lines were used for these studies: (1) MCF-10F cells, a normal human breast epithelial cell line, and (2) MCF-7 cells, a human breast adenocarcinoma cell line. Both cell lines were stained with the dye aluminum phthalocyanine tetrasulfonate (APT) and prepared for spectroscopic study at liquid helium temperatures. Routinely measured hole burning parameters were obtained for the two cell types and compared in order to differentiate between physical properties of normal and cancer cells;A comparison of the fluorescence excitation spectra for APT in the suspension media and suspensions of MCF-10F and MCF-7 cells stained with APT indicate that the dye has successfully been internalized into the cells. Holes were burned into the cell samples and the mechanism was shown to be nonphotochemical, meaning that the intracellular environments of MCF-10F and MCF-7 cells are amorphous at 4.5 K. The fluorescence excitation spectra and hole-burned spectra for APT in cells were compared to APT in hyperquenched glassy films of water, ethanol, and methanol. Results for APT in MCF-10F cells show that the dye is in an acidic, aqueous environment with a hydrogen-bonded network similar to that of amorphous water, but exhibiting influences form other intracellular species. Pressure induced shifting of holes allowed for determination of local compressibilities (ca. 0.06 GPa-1) about APT molecules for both cell types. External electric (Stark) fields were applied to MCF-10F and MCF-7 cells stained with APT. Stark fields resulted in hole broadening for both cell lines when laser polarization was parallel and perpendicular to the applied electric field. Stark hole burning was able to distinguish between normal and cancer cells based on changes in the dipole moments for APT in MCF-10F and MCF-7 cells. Differences in the change in dipole moment suggest that dye molecules near the plasma membrane of normal cells experience ordering not found in cancer cells.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Milanovich, Nebojsa, "Application of spectral hole burning to the study of in vitro cellular systems " (1999). Retrospective Theses and Dissertations. 12594.