Identification of site-specific isotopic labels by vibrational spectroscopy in the electron microscope.

The identification of isotopic labels by conventional macroscopic techniques lacks spatial resolution and requires relatively large quantities of material for measurements. We recorded the vibrational spectra of an α amino acid, l-alanine, with damage-free "aloof" electron energy-loss spectroscopy in a scanning transmission electron microscope to directly resolve carbon-site-specific isotopic labels in real space with nanoscale spatial resolution. An isotopic red shift of 4.8 ± 0.4 milli-electron volts in C-O asymmetric stretching modes was observed for C-labeled l-alanine at the carboxylate carbon site, which was confirmed by macroscopic infrared spectroscopy and theoretical calculations. The accurate measurement of this shift opens the door to nondestructive, site-specific, spatially resolved identification of isotopically labeled molecules with the electron microscope.