FTIR Spectroscopy Demonstrates Biochemical Differences in Mammalian Cell Cultures at Different Growth Stages

Abstract

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.

Growth substrate induced functional changes elucidated by FTIR and Raman spectroscopy in in–vitro cultured human keratinocytes

“…This difficulty may be lessened through careful choice of spectral intervals for integration, as was the approach adopted here. The spectral changes observed here are consistent with those previously observed in experiments utilising spectral decomposition [8], and as such support the approach adopted here. All measurements are normalised to the corresponding control measurement.…”

“…within the spectrum [8]. This difficulty may be lessened through careful choice of spectral intervals for integration, as was the approach adopted here.…”

“…At present Fourier Transform Infrared Microspectrosopy (FTIRM) and Confocal Raman Microspectroscopy (CRM) have identified in-situ molecular alterations associated with cell death via apoptosis [1][2][3] or necrosis [4,5], or as a result of proliferative changes in cellular activity [6][7][8] or mitosis [9]. It has also been shown that these modalities can identify molecular changes associated with changes in the phenotype of differentiating embryonic cells [10,11].…”

Discrimination between healthy and tumor tissues on formalin-fixed paraffin-embedded breast cancer samples using IR imaging

“…Yet most of these differences were located in the DNA/RNA spectral region. Several studies have previously reported spectral differences for cells at different stages of the eukaryotic cell division cycle [1,2,13]. These differences are caused by the morphological and biochemical changes occurring within the cell division cycle.…”

“…Otherwise, the nucleic acid contribution of the spectrum is mostly due to cytoplasmic RNA, DNA being too tightly packed in the nucleus to allow the infrared radiation to cross through [1]. Meanwhile, Mourant et al demonstrated that the ratio RNA/lipid could be used to determine the proliferative status of mammalian cells [13]. A diagnostic advantage of the FT-IR spectroscopy in cancer research is that the development of a cancer DNA phenotype can be observed prior to the tumor formation, notably for prostate carcinomas [11].…”