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.

[1]  Christoph Krafft,et al.  Mapping of single cells by near infrared Raman microspectroscopy , 2003 .

[2]  J. Greve,et al.  Carotenoids located in human lymphocyte subpopulations and natural killer cells by Raman microspectroscopy. , 1993, Cytometry.

[3]  W. R. Wiley,et al.  Three-Dimensional Vibrational Imaging by Coherent Anti-Stokes Raman Scattering , 1999 .

[4]  B. Wood,et al.  Micro-Raman characterisation of the R to T state transition of haemoglobin within a single living erythrocyte. , 2001, Biochimica et biophysica acta.

[5]  Nicholas R Cozzarelli,et al.  Comparison of recombination in vitro and in E. coli cells: Measure of the effective concentr ation of DNA in vivo , 1995, Cell.

[6]  Christoph Krafft,et al.  Near-infrared Raman spectroscopy to study the composition of human brain tissue and tumors , 2003, European Conference on Biomedical Optics.

[7]  T. Lindmo,et al.  Flow cytometry and sorting , 1979 .

[8]  Andreas Volkmer,et al.  Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy and Study of Lipid Vesicles , 2002 .

[9]  P. Treado,et al.  Peer reviewed: Raman chemical imaging spectroscopy. , 1999, Analytical chemistry.

[10]  Michael S. Feld,et al.  Surface-Enhanced Raman Spectroscopy in Single Living Cells Using Gold Nanoparticles , 2002 .

[11]  B Willekens,et al.  Nonresonant Raman imaging of protein distribution in single human cells. , 2003, Biopolymers.

[12]  J Greve,et al.  Nonresonant confocal Raman imaging of DNA and protein distribution in apoptotic cells. , 2003, Biophysical journal.

[13]  D. Borchman,et al.  Lipid composition, membrane structure relationships in lens and muscle sarcoplasmic reticulum membranes. , 1999, Biospectroscopy.