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Isotope labeling is a fundamental staple for the study of cellular metabolismand protein function. The conventional techniques that allow resolution andidentification of isotopically-labeled biomarkers, such as mass spectrometryand infrared spectroscopy, are macroscopic in nature and have the disadvantageof requiring relatively large quantities of material and lacking spatialresolution. Here, we record the vibrational spectra of an {\alpha}-amino acid,L-alanine, using spatially-resolved monochromated electron energy lossspectroscopy (EELS) to directly resolve carbon-site-specific isotopic labels ina scanning transmission electron microscope. The EELS is acquired in aloofmode, meaning the probe is positioned away from the sample (~20 nm) sparing thesensitive biomolecule from the high-energy excitations, while the vibrationalmodes are investigated. An isotopic red-shift of 5.3 meV was obtained for theC=O stretching mode in the carboxylic acid group for 13C-enriched L-alaninewhen compared with naturally occurring 12C L-alanine, which is confirmed bymacroscopic infrared spectroscopy measurements and theoretical calculations.The EELS experiments presented here are the first demonstration ofnon-destructive resolution and identification of isotopically-labeled aminoacids in the electron microscope, opening a new door for the study ofbiological matter at the nanoscale.

Seeing Site-Specific Isotopic Labeling of Amino Acids with Vibrational Spectroscopy in the Electron Microscope