• biomolecular component analysis;
  • nucleolus;
  • site-specific Raman microspetrometry;
  • principal component analysis

Distinct cellular domains, such as structure–function compartments of the cell nucleus and cytoplasmic organelles, are responsible for numerous macromolecular processes essential for cell functions. Spectroscopic analysis of specific cellular domains opens a way for noninvasive characterization of their molecular content and monitoring of their function.

Confocal Raman spectroscopy was employed here for characterization of the complex molecular organization of major structure–function compartment of the cell nucleus, the nucleolus. The Raman spectra obtained in the nucleoli were processed by biomolecular component analysis (BCA). BCA was used to determine the contribution of each major type of macromolecules (proteins, DNA, RNA and lipids) to the complex molecular composition of nucleoli. A notable cell-to-cell variability in the macromolecular composition of nucleolus was found. At the same time, we observed a correlation between the concentrations of major types of biomolecules in this nuclear compartment. In particular, the averaged concentration of RNA increases along with increase in protein concentration, while an inverse dependence between the concentrations of RNA and DNA was found. Variations in the nucleolar concentrations of lipids were also noticed. Manifestations in spectral variations of proteins for individual nucleoli, shown by BCA, are discussed and interpreted.

We also compared utility of BCA and principal component analysis for biomolecular studies and conclude that BCA is a more powerful and informative technique for studies of macromolecular composition and its variations in specific subcellular domains. Copyright © 2012 John Wiley & Sons, Ltd.