The new electron microscopy offers at the nanoscale, d


image: Electrifying neutrons: Monochromatic electron energy loss spectroscopy under a scanning transmission electron microscope is used to distinguish molecules that differ only by a single neutron on a single atom. The electron beam can capture changes in tiny molecular vibrations of an amino acid caused by the extra neutron without damaging the sample and with unprecedented spatial resolution.
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Credit: Andy Sproles / Oak Ridge National Laboratory, US Dept. of Energy

OAK RIDGE, Tenn., January 31, 2019 – A new electron microscopy technique that detects subtle changes in protein weight at the nanoscale – while keeping the sample intact – could open a new avenue for research deeper and more comprehensive studies of the basics of life.

Scientists at the Department of Energy’s Oak Ridge National Laboratory described in the newspaper Science the first use of an electron microscope to directly identify isotopes in amino acids at the nanoscale without damaging the samples.

Isotopes are commonly used to label molecules and proteins. By measuring changes in the vibrational signatures of the molecule, the electron microscope can track isotopes with unprecedented spectral precision and spatial resolution.

The technique does not destroy amino acids, allowing real-space observation of dynamic chemistry and creating a basis for a multitude of scientific discoveries ranging from simple to complex biological structures in the life sciences.

“The way we understand disease progression, human metabolism and other complex biological phenomena is based on interactions between proteins,” said Jordan Hachtel, ORNL postdoctoral fellow and lead author. “We study these interactions by tagging specific proteins with an isotope and then following them through a chemical reaction to see where they went and what they did.”

“Now we can follow isotopic markers directly with the electron microscope, which means we can do it with spatial resolution comparable to the actual size of proteins,” Hachtel added.

Their new experiment, which took place at ORNL’s Center for Nanophase Materials Science, used monochromatic electron energy loss spectroscopy, or EELS, in a scanning transmission electron microscope, or STEM. The technique used by scientists is sensitive enough to distinguish molecules that differ by a single neutron on a single atom. EELS has been used to capture minute vibrations in the molecular structure of an amino acid.

“Isotopic markers are typically observed at the macroscopic level using mass spectrometry, a scientific tool that reveals the atomic weight and isotopic composition of a sample,” said Juan Carlos Idrobo, ORNL scientist and corresponding author. “Mass spectrometry has incredible mass resolution, but it usually doesn’t have nanometric spatial resolution. It’s a destructive technique.”

A mass spectrometer uses an electron beam to separate a molecule into charged fragments which are then characterized by their mass / charge ratio. By observing the sample at the macroscopic scale, scientists can only statistically infer the chemical bonds that may have existed in the sample. The sample is destroyed during the experiment, leaving valuable information undiscovered.

The new electron microscopy technique, as applied by the ORNL team, offers a smoother approach. By positioning the electron beam extremely close to the sample, but not directly touching it, the electrons can excite and sense vibrations without destroying the sample, allowing observations of biological samples at room temperature over longer periods of time. .

Their result is a breakthrough for electron microscopy, since the negatively charged electron beam is generally only sensitive to protons, not neutrons. “However, the frequency of molecular vibrations depends on atomic weight, and the precise measurement of these vibrational frequencies opens the first direct channel for measuring isotopes under an electron microscope,” Idrobo said.

The ORNL-led research team expects that their potentially revolutionary technology will not replace, but rather complement, mass spectrometry and other conventional optical and neutron techniques currently used to detect isotopic markers.

“Our technique is the perfect complement to a large-scale mass spectrometry experiment,” said Hachtel. “With the pre-knowledge of mass spectrometry, we can enter and spatially resolve where isotopic markers are found in a real space sample.”

Beyond life sciences, the technique could be applied to other soft materials such as polymers, and potentially in quantum materials where isotope substitution can play a key role in the control of superconductivity.

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The co-authors of the study entitled “Identification of Site-Specific Isotopic Labels by Vibrational Spectroscopy in the Electron Microscope”, included Jordan A. Hachtel, Jingsong Huang, Ilja Popovs, Santa Jansone-Popova, Jong K. Keum, Jacek Jakowski and Juan Carlos Idrobo, all of ORNL, and Tracy C. Lovejoy, Niklas Dellby and Ondrej L. Krivanek of Nion Co., the designers and manufacturers of the electron microscope and spectrometer used in the experiments.

The research was supported by the DOE Bureau of Science and used an aberration corrected monochrome scanning transmission electron microscope, or MAC-STEM, and other resources from the Center for Nanophase Materials Sciences of the ORNL, a user installation of DOE.

UT-Battelle manages ORNL for the DOE Science Office. The Office of Science is the largest supporter of basic research in the physical sciences in the United States and works to address some of the most pressing challenges of our time. For more information, please visit science.energy.gov.


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