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.

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).

Infrared (IR) spectroscopy of biological cells is a growing area of research, with many papers focusing on differences between the spectra of cancerous and noncancerous cells. Much of this research has been performed using a monolayer of dehydrated cells. We posit that the use of monolayers can introduce artefacts that lead to an apparent but inaccurate measurement of differences between cancerous and noncancerous cells. Additionally, the use of dried cells complicates the extraction of biochemical information from the IR spectra. We demonstrate that using suspensions of viable cells in aqueous suspension reduces measurement artefacts and facilitates determining the concentration of the major biochemical components via a linear least-squares fit of the component spectra to the spectrum of the cells.

The ratio of nonresonant two-photon induced fluorescence excitation spectra using circularly and linearly polarized light for jet-cooled 3-methylindole complexed with a series of increasingly basic hydrogen bond acceptors (water, methanol, ethanol, diethylether, diethylamine and triethylamine) is consistent with an avoided crossing of the two lowest excited singlet states, La1 and Lb1. The dispersed fluorescence of these from this series also reflects the crossing, providing a definitive La1 jet-cooled fluorescence spectrum. The jet-cooled La1 fluorescence spectrum is not broad and redshifted, but has vibronic structure that agrees well with ab initio predictions and is similar to that of La3 phosphorescence.

Two-photon polarized fluorescence excitation and vibronically resolved one-photon dispersed fluorescence spectra of the long-wavelength absorbing van der Waals complexes of indole with water, methanol, and formamide were examined for the purpose of firmly assigning the nature of the lowest singlet excited electronic state. The two-photon spectra for all of these complexes have circular/linear polarization ratios of absorptivity (Ω-values) that show that excitation is to the Lb1 state. Analysis of the Franck–Condon (FC) patterns of the dispersed fluorescence for these indole-polar solvent complexes show that emission is also from the Lb1 state in each case. In the emission spectra, the intensity ratios of the origin and ν26 lines are about 2:1, which is the value expected for Lb1 emission. The ν26 vibration is the most intense nonorigin vibronic line, as expected for Lb1 emission. Finally, there is little or no intensity from the ν8, ν9, and ν10 vibrations, which would be strongly active with emission from the La1 state. These results show unequivocally that for these indole-polar solvent complexes the La1 state does not shift in energy below the Lb1 state—contrary to the interpretation of several recent papers. Further support is given to the assignment of Lb1 emission by spectral simulations. The jet-cooled complexes do not exhibit excited state complex (exciplex) characteristics, wherein the solvent is much more strongly bound in the excited state than in the ground state.