Identification of organelles and vesicles in single cells by Raman microspectroscopic mapping

Scope of the present paper is to develop non-resonant Raman microspectroscopic mapping techniques for single cells studies in order to identify subcellular structures by their spectral signatures. Human lung fibroblast cells were fixed in formalin and stored in buffer in order to prevent morphological and chemical changes during data acquisition. Raman maps were recorded with 1 μm step size at 785 nm excitation and with 0.3 μm step size at 532 nm excitation. Spectral details could be resolved such as the distinction of RNA and DNA, proteins, cholesterol and phospholipids like phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Fitting of cluster averaged spectra by linear combinations of reference spectra was used to approximate the subcellular composition. Images could be reconstructed revealing the protein, nucleic acid and lipid concentration. Based on these compositional information, color coded cluster memberships were correlated with nucleus, cytoplasm, endoplasmic reticulum, vesicles and peripheral membrane.