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

TL;DR

This paper demonstrates a novel, non-destructive method using electron energy loss spectroscopy to resolve site-specific isotopic labels in amino acids at the nanoscale, surpassing traditional macroscopic techniques.

Contribution

It introduces the first use of vibrational EELS in a scanning transmission electron microscope for site-specific isotopic labeling of amino acids, enabling nanoscale biological studies.

Findings

Resolved carbon isotopic shifts in amino acids using EELS

Confirmed vibrational shifts with infrared spectroscopy and calculations

Achieved non-destructive, spatially-resolved isotopic detection in biological molecules

Abstract

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