title: Noninvasive imaging of protein metabolic labeling in single human cells using stable isotopes and Raman microscopy.
abstract: We have combined nonresonant Raman microspectroscopy and spectral imaging with stable isotope labeling by amino acids in cell culture (SILAC) to selectively detect the incorporation of deuterium-labeled phenylalanine, tyrosine, and methionine into proteins in intact, single HeLa cells. The C-D stretching vibrational bands in these amino acids are observed in the 2100-2300 cm(-1) spectral region that is devoid of vibrational contributions from other, nondeuterated intracellular constituents. We found that incubation with deuterated amino acids for 8 h in cell culture already led to clearly detectable isotope-related signals in Raman spectra of HeLa cells. As expected, the level of isotope incorporation into proteins increased with incubation time, reaching 55% for deuterated phenylalanine after 28 h. Raman spectral imaging of HeLa cells incubated with deuterium-labeled amino acids showed similar spatial distributions for both isotope-labeled and unlabeled proteins, as evidenced by Raman ratio imaging. The SILAC-Raman methodology presented here combines the strengths of stable isotopic labeling of cells with the nondestructive and quantitative nature of Raman chemical imaging and is likely to become a powerful tool in both cell biology applications and research on tissues or whole organisms.
authors.affiliation: Biophysical Engineering Group, MESA+ Institute for Nanotechnology, University of Twente, P.O. Box 217, 7500 AE Enschede, The Netherlands.
authors.forename: Henk-Jan
authors.initials: HJ
authors.lastname: van Manen
authors.forename: Aufried
authors.initials: A
authors.lastname: Lenferink
authors.forename: Cees
authors.initials: C
authors.lastname: Otto
mesh_terms: D003903:Deuterium / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D008715:Methionine / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D011506:Proteins; D014443:Tyrosine; D010649:Phenylalanine; D008715:Methionine; D003903:Deuterium