Two-photon photodynamic therapy has the advantages of being highly localized in its effects and allows for deeper
tissue penetration, when compared to one-photon photodynamic therapy. N-alkylated 3,5-bis(arylidene)-4-piperidones,
with a donor-pi-acceptor-pi-donor structure, have the potential to be useful two-photon sensitizers. We have measured
two-photon cross sections (using femtosecond excitation), fluorescence quantum yields, fluorescence lifetimes, and xray
crystal structures for a number of these compounds. Most two-photon cross sections are comparable to or larger than
that of Rhodamine B. However, the fluorescence quantum yields are low (all less than 10%) and the fluorescence
lifetimes are less than 1 ns (with one exception), suggesting that there may be a significant energy transfer to the triplet
state. This would encourage singlet oxygen formation and increase cellular toxicity. Results of dark cyto-toxicity studies
with several human cancer cell lines are presented. White light photo-toxicity results are also presented, and suggest that
increasing the number of double bonds, from one to two, in the piperidone wings increases the photo-toxicity with little
corresponding change in the dark cyto-toxicity.
We have conducted an extensive comparison of cellular biochemical composition obtained from infrared and Raman spectra of intact cells with measurements using standard extraction and chemical analysis (including NMR), and flow cytometric assay on fixed cells. Measurements were conducted on a rat fibroblast carcinogenesis model consisting of normal and tumorigenic cells assayed as exponentially growing and plateau-phase cultures. Estimates of protein, DNA, RNA, lipids, and glycogen amounts were obtained from a previous publication in which vibrational spectra were fit to a set of basis spectra representing protein, DNA, RNA, lipids, and glycogen. The Raman spectral estimates of absolute cellular composition were quite similar to the independent biochemical and flow cytometric assays. The infrared spectra gave similar results for protein, lipid, and glycogen but underestimated the DNA content while overestimating the RNA level. When ratios of biochemical concentrations in exponential and plateau-phase cultures were examined, the Raman spectroscopic results were the same, within errors, as the independent methods, in all cases. Several changes in relative biochemical composition due to tumorigenic and proliferative status previously reported using vibrational spectroscopy were confirmed by the independent methods. These results demonstrate that vibrational spectroscopy can provide reliable estimates of the biochemical composition of mammalian cells.
The biochemical composition of mammalian cells has been estimated by Raman spectroscopy and the results compared with other biochemical methods. The Raman spectroscopy estimates were performed by fitting measured Raman and infrared spectra of dense cell suspensions to a linear combination of basis components (RNA, DNA, protein, lipid, glycoen). The Raman spectroscopy results are compared to biochemical analyses performed by extraction and quantfication of the biochemical components. Both absolute and relative measurements of biochemical
composition are compared. Both the Raman and biochemical results indicate that there are signficant differences in gross biochemical composition dependent on growth stage and tumorigneicity.
Both infrared and Raman spectroscopies have the potential to noninvasively estimate the biochemical composition of mammalian cells, although this cannot be unambiguously determined from analysis approaches such as peak assignment or multivariate classification methods. We have developed a fitting routine that determines biochemical composition using basis spectra for the major types of biochemicals found in mammalian cells (protein, DNA, RNA, lipid and glycogen), which is shown to be robust and reproducible. We measured both infrared and Raman spectra of viable suspensions of pairs of nontumorigenic and tumorigenic rat fibroblast cell lines. To model in vivo conditions, we compared nonproliferating, nontumorigenic cells to proliferating, tumorigenic cells. Reproducible differences in biochemical composition were found for both nontumorigenic/tumorigenic cell models, using both spectroscopic techniques. These included an increased fraction of protein and nucleic acids in the tumorigenic cells, with a corresponding decrease in lipid and glycogen fractions. Measurements of each cell type in both the proliferating and nonproliferating states showed that proliferative status was the major determinant of differences in vibrational spectra, rather than tumorigenicity per se. The smallness of the spectral changes associated with tumorgenicity may be due to the subtle nature of the oncogenic change in this system (a single mutant oncogene).
We have developed methods of measuring absorption of transient species utilizing stepped-scan Fourier transform interferometry that allows a combination of broad spectral coverage (10,000 - 15,000 cm-1 per spectrum), good spectral resolution, and up to ns temporal resolution with possibilities of extension to the ps domain. Nanosecond, psec or fsec laser systems, tunable from UV to IR can be used as the pump source to prepare the transient species. The absorption of that species is measured with broadband, incoherent light and can be simultaneously time and frequency resolved.
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