(534b) A New Type of Electron Microscopy Experiment for Biological Specimens: Vibrational Electron Energy Loss Spectroscopy

Electron microscopy has played a critical role in the characterization of biological samples over the last half-century. Much of this work has centered around cryogenic single-particle-analysis in bright field transmission electron microscopy (TEM), yet TEM’s complementary counterpart, scanning TEM (STEM)¹, is far less frequently used due to the high beam damage associated with STEM’s converged probe. However, the critical advantage of STEM is access to localized spectroscopy with the converged probe. Electron energy-loss spectroscopy (EELS) in particular has seen use in biological materials due to its ability to provide element-specific information from the STEM probe².

Recently, a new opportunity has emerged in STEM-EELS due to advancements in monochromation³. The native resolution of an EELS measurement is traditionally governed by the spread of energies that emerge from the gun, which for the best field emission sources is approximately 300 meV (2400 cm^-1). However, by monochromating this resolution can be improved by two orders of magnitude, and now energy resolutions of 6-8 meV (50-60 cm^-1) are readily achievable, and resolutions as high 2-3 meV (15-25 cm^-1) achievable in special circumstances^4,5. Most critically, these advances are achieved without reducing the spatial resolution of the electron gun, enabling vibrational spectroscopy sensitive to individual atomic vibrations⁶.

The access to vibrations has also had an additional benefit towards biological materials, which is the advent of ‘aloof’ EELS. Where the beam is placed close (but not intersecting) with the sample, and the vibrational response is measured through the evanescent beam-sample interactions. The strength of this aloof effect is inversely proportional to the excitation frequency, so low-energy infrared vibrational modes (which do not harm the sample) are excited, but higher energy visible/UV electronic excitations (which break apart bonds) are not excited⁷. As a result, the electron beam can be placed 20-30 nm away from the sample and can still be used to conduct nanoscale vibrational spectroscopy measurements without incurring beam damage⁸.

In this talk, I will show three different ways we can apply monochromated EELS to perform nanoscale vibrational spectroscopy experiments on biological/biology-adjacent materials. I will demonstrate how we can use the aloof effect to perform aloof linescans and distinguish between amino acids with different isotopic labels in real-space⁹, I will show how we can use careful confinement of water into liquid cells/carbon nanotubes to unveil the structure of liquids with vibrational EELS, and I will show how we can move past aloof EELS to conduct real-space vibrational analysis of beam-sensitive whole-cell structures and still retain ~15 nm spatial resolution and understand the resulting beam-damage effects.

References

1. Henderson, R. & Unwin, P. N. T. Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257, 28–32 (1975).
2. Aronova, M. A. & Leapman, R. D. Development of electron energy-loss spectroscopy in the biological sciences. MRS Bulletin 87, 53 (2012).
3. Krivanek, O. L. et al. Vibrational spectroscopy in the electron microscope. Nature 514, 209–212 (2014).
4. Hachtel, J. A., Lupini, A. R. & Idrobo, J. C. Exploring the capabilities of monochromated electron energy loss spectroscopy in the infrared regime. Scientific Reports 8, 5637 (2018).
5. Dellby, N. et al. Ultra-high Resolution EELS Analysis and STEM Imaging at 20 keV. Microscopy and Microanalysis 29, 626–627 (2023).
6. Xu, M. et al. Single-atom vibrational spectroscopy with chemical-bonding sensitivity. Nat. Mater. 22, 612–618 (2023).
7. Ercius, P., Hachtel, J. A. & Klie, R. F. Chemical and bonding analysis of liquids using liquid cell electron microscopy. MRS Bulletin 45, 761–768 (2020).
8. Rez, P. et al. Damage-free vibrational spectroscopy of biological materials in the electron microscope. Nature Communications 7, 10945 (2016).
9. Hachtel, J. A. et al. Identification of site-specific isotopic labels by vibrational spectroscopy in the electron microscope. Science 363, 525–528 (2019).