Carcinoma and SV40-Transfected Normal Ovarian Surface Epithelial Cell Comparison by Nonphotochemical Hole Burning

Abstract

Results are presented of nonphotochemical-hole-burning experiments on the mitochondrial specific dye rhodamine 800 incubated with two human ovarian surface epithelial cell lines: OSE(tsT)-14 normal cells and OV167 carcinoma cells. This dye is selective for the plasma and inner membranes of the mitochondria, as shown by confocal microscopy images. Dispersive hole-growth kinetics of zero-phonon holes are analyzed with theoretical fits, indicating that subcellular structural heterogeneity of the carcinoma cell line is lower relative to the analogous normal cell line. Broadening of holes in the presence of an applied electric field (Stark effect) was used to determine the permanent dipole moment change for the S(0)-->S(1) transition in the two cell lines. For the carcinoma cell line, the permanent dipole moment change value is a factor of 1.5 higher than for the normal cell line. It is speculated that this difference may be related to differences in mitochondrial membrane potentials in the two cell lines.

Nonphotochemical Hole-Burning Study of Selectively Stained Normal and Cancerous Human Ovarian Tissues

“…A similar conclusion was reached in refs and , where further advances were made with significant differences measured in the areas of burn kinetics and the Stark effect, further suggesting that NPHB has potential as an imaging technique used to reveal subcellular anomalies. For example, a significant difference in the dipole moment change (Stark effect) and different hole-growth kinetics of the zero-phonon hole (ZPH) were found between the OV167 carcinoma (cancer) cells and the OSE(tsT)-14 cells. , That is, the width of the ZPH was dependent on an applied electric field ( E St ) with laser excitation ( E L ) either parallel or perpendicular to E St . This dependence yielded the permanent dipole moment change associated with the S 0 → S 1 optical transition of MF680.…”

“…The PSBH is observed to be 24 ± 4 cm -1 for each tissue sample with no significant differences in the vibronic satellite hole structures. The latter value is smaller by ∼6 cm -1 than that observed in cells . The most noticeable vibronic peaks on both sides of ZPH are at 227 cm -1 as indicated by the arrows.…”

“…The absorption and emission maxima in ethanol were measured at 682 and 702 nm, respectively, while the absorption maximum of MF680 in HGW (Figure A, curve d) is near 672 nm. None of these matrixes have any lipid nature whatsoever; in each tissue sample, however, the dye molecule is lipophilic and cationic and, as shown in ref , is preferentially located in the lipid-based (negatively charged) mitochondria. Another factor was elucidated in the work presented by Sakanoue et al, who found that on isolated rat liver mitochondria the MF680 dye molecule displayed spectral changes dependent on ΔΨ m .…”

“…One notable feature of the image is the specific highlights of the dye in structured organelles, which most closely resemble mitochondria when compared to other studies dealing with the staining of in situ mitochondria . The sodium azide test described in ref was conducted to rule out the possibility that the MF680 dye was locating preferentially somewhere other than the mitochondria or even entrapped in the intercellular matrixes with this concentration. Briefly, a small amount of sodium azide was added to the sample in PBS while it was being imaged on the microscope.…”

“…Nonphotochemical hole-burning (NPHB) of numerous dye molecules in various polymers and glasses has been extensively studied to show that NPHB is sensitive to the microenvironment around the dye. − Hole-burning imaging (HBI), a technique that exploits the inherent nano-environmental sensitivity of NPHB for detecting quantifiable differences between cancerous and analogous normal cells, has been the subject of recent papers. , The procedural feasibility of HBI as a cancer diagnostic tool was explored in refs and , where it was shown that NPHB of intracellular dye molecules could be used to reveal anomalies in subcellular structures. The above studies used two distinct cultured human ovarian surface epithelial cell linesOV167 carcinoma and OSE(tsT)-14 temperature-sensitive SV40 transfected normal cellsboth stained with the mitochondria-specific dye, Molecular Probes MitoFluor Far Red 680 (MF680), commonly known as rhodamine 800.…”