Raman spectra of cells and nuclei from cultures in the plateau (nonproliferating) and exponential (proliferating) phases of growth were measured and show that Raman spectroscopy can monitor changes due to cell proliferation. A simple fitting routine was developed using a basis set (lipid, protein, DNA, RNA) to estimate the relative amounts of biochemical components in cells and nuclei. Using relative amounts and ratios of biochemical components, reproducible differences can be detected and quantified that are not readily apparent by visual analysis of vibrational bands in the spectra. These differences, due to cell proliferation, can be assigned to specific biochemical changes. They include a decrease in the relative lipid and increases in the relative protein and RNA for both nontumorigenic exponential cells and nuclei, and an increase in the relative RNA for tumorigenic exponential cells. The lipid/RNA ratio decreases for nontumorigenic exponential cells and nuclei and tumorigenic exponential cells. The protein/lipid ratio increases for both tumorigenic and nontumorigenic exponential cells and nuclei. Finally, the lipid/DNA ratio decreases for tumorigenic exponential nuclei. This knowledge will be important for Raman detection of rapidly dividing populations of cancer cells in vivo.

We have observed differences in the infrared spectra of viable fibroblast cells depending on whether the cells were in the exponential (proliferating) or plateau (nonproliferating) phase of growth. The spectral changes were observed even after correcting for cell number and volume, ruling out these trivial explanations. Several of the changes occurred for both transformed and normal cell lines and were greater for the normal cell line. The biochemical basis of the spectral changes was estimated by fitting the cell spectra to a linear superposition of spectra for the major biochemical components of mammalian cells (DNA, RNA, protein, lipids, and glycogen). The ratios of RNA/lipid and protein/lipid increased when the cells were in the exponential phase compared to the plateau phase of growth. The fits of cell spectra to individual biochemical components also demonstrated that DNA is a relatively minor spectroscopic component as would be expected biochemically. Contrary to other reports in the literature, our data demonstrate that determining DNA content or structure using Fourier transform infrared spectroscopy data is difficult due to the relatively small amount of DNA and the overlap of DNA bands with the absorption bands of other biochemical components.

An understanding of the relationship between tissue structures and light scattering from tissue will help facilitate the development and acceptance of noninvasive optical diagnostics including elastic scattering spectroscopy, diffuse reflectance, and optical coherence tomography. For example, a quantitative model of the structures that scatter light in epithelial cells would allow determination of what structures control the characteristics of in vivo light transport measurements and subsequently could provide a detailed relationship between cellular structures and optical measurements. We have determined the size distribution of refractive index structure variations in epithelial cells as well as in nuclei isolated from epithelial cells from measurements of the angular dependence of polarized light scattering. The quantitative size distributions we obtained for both whole cells and isolated nuclei include particles with effective radii of 2 microm to 10 nm or less and contain orders of magnitude more small particles than large particles. These results demonstrate that not only are biological cells very heterogeneous, but so are the nuclei within them. Light scattering is likely sensitive to structures smaller than those commonly investigated by standard pathology methods.

Cell inactivation after exposure to collimated 3.5-MeV alpha particles in three hamster cell lines, V79, CHO-10B, and HS-23, one mouse cell line, C3H 10T1/2, and a human skin fibroblast cell line were studied. Several parameters were investigated for each cell line. Theoretical calculations were performed to find the distribution of energy deposited in the nuclear volume for each cell line. The mean number of alpha-particle traversals required to induce a lethal lesion varied between two for HS-23 cells and six for C3H 10T1/2 cells. The number of traversals per unit area and the total track length of alpha particles that inactivated a cell were found to be nearly constant for the hamster and mouse cell lines. These quantities were found to be lower for the human skin fibroblast cell line. The RBE values for all cell lines were found to be about 3.8 at 10% survival. Thus cell lines that are more sensitive to alpha radiation are also more sensitive to gamma radiation. The average number of alpha-particle traversals producing a single lethal lesion is greater than one. The passages of alpha particles through the cell nucleus that do not kill the cell may lead to carcinogenic effects.

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).