Hole Burning Imaging Studies of Cancerous and Analogous Normal Ovarian Tissues Utilizing Organelle Specific Dyes

摘要

Presented in this dissertation is the successful demonstration that nonphotochemical hole burning (NPWB) imaging can be used to study in vitro tissue cellular systems for discerning differences in cellular ultrastructures due to cancer development. This has been accomplished with the surgically removed cancerous ovarian and analogous normal peritoneal tissues from the same patient and the application of a fluorescent mitochondrion specific dye, Molecular Probe MitoFluor Far Red 680 (MF680), commonly known as rhodamine 800, that has been proven to exhibit efficient NPHB. From the results presented in Chapters 4 and 5 , and Appendix B, the following conclusions were made: (1) fluorescence excitation spectra of MF680 and confocal microscopy images of thin sliced tissues incubated with MF680 confirm the site-specificity of the probe molecules in the cellular systems. (2) Tunneling parameters, (lambda)(sub 0) and (sigma)(sub (lambda)), as well as the standard hole burning parameters (namely, (gamma) and S), have been determined for the tissue samples by hole growth kinetics (HGK) analyses. Unlike the preliminary cultured cell studies, these parameters have not shown the ability to distinguish tissue cellular matrices surrounding the chromophores. (3) Effects of an external electric (Stark) field on the nonphotochemical holes have been used to determine the changes in permanent dipole moment (f(Delta)(mu)) for MF680 in tissue samples when burn laser polarization is parallel to the Stark field. Differences are detected between f(Delta)(mu)s in the two tissue samples, with the cancerous tissue exhibiting a more pronounced change (1.35-fold increase) in permanent dipole moment change relative to the normal analogs. It is speculated that the difference may be related to differences in mitochondrial membrane potentials in these tissue samples. (4) In the HGK mode, hole burning imaging (HBI) of cells adhered to coverslips and cooled to liquid helium temperatures in the complete absence of cryopreservatives, shows the ability to distinguish between carcinoma and analogous normal cells on the single-cell level. In future applications, this system has the potential to be used with smears of tissue samples for single-layer HBI analysis. These conclusions demonstrate that HBI has the potential of providing detailed information about localized intracellular environments and for detecting changes in the physical characteristics (e.g., electrical properties) of cells which constitute the in vitro tissue samples. For the latter, the long-term goal will be to develop NPHB into a diagnostic technique for the early detection of cancer by exploiting the physical differences between normal and cancerous cells and tissues. Moreover, because of the aforementioned HBI's capability to detect cellular anomalies, it has the potential of being used in conjunction with studies involving photodynamic therapy, assuming the chromophore is carefully selected.