Damage-free vibrational spectroscopy of biological materials in the electron microscope

Vibrational spectroscopy in the electron microscope would be transformative in the study of biological samples, provided that radiation damage could be prevented. However, electron beams typically create high-energy excitations that severely accelerate sample degradation. Here this major difficulty is overcome using an ‘aloof' electron beam, positioned tens of nanometres away from the sample: high-energy excitations are suppressed, while vibrational modes of energies <1 eV can be ‘safely' investigated. To demonstrate the potential of aloof spectroscopy, we record electron energy loss spectra from biogenic guanine crystals in their native state, resolving their characteristic C–H, N–H and C=O vibrational signatures with no observable radiation damage. The technique opens up the possibility of non-damaging compositional analyses of organic functional groups, including non-crystalline biological materials, at a spatial resolution of ∼10 nm, simultaneously combined with imaging in the electron microscope.


Supplementary Figure 2 Variation of UV Region Spectra with Electron Probe Position
EEL Spectra in the visible and UV region as the beam is moved closer to the specimen.
Supplementary Figure 2 shows the EELS signal in the visible and UV region as the beam is moved closer and into the sample. The positions for the spectra are marked in the image. Note that the peak at 7 eV, only appears when the beam is inside the specimen. The ratio of the heights of the 4.04 eV and 6 eV peaks changes as the beam moves into the specimen and the background from the  plasmon becomes more apparent. The fluence needed to reduce the intensity by 1/e is 0.5 e /nm 2 which is equivalent to a dose of 8 x10 -6 C/cm 2 . This is much lower than the fluence quoted by Siangchaew and Libera for polystyrene which was measured from changes in the  peak intensity 3,4 It is also lower than the equivalent fluence published by Li and Egerton 5 for various aromatic compounds measured from both changes in the  peak intensity and reduction in the intensity of the strongest diffraction spot. This is no doubt due to the higher order reflection being more sensitive to high resolution structural information that is strongly affected by displacements of the guanine molecules within the unit cell. Supplementary Figure 5a shows the raw spectra, Supplementary Figure 5b shows the spectra after background subtraction using the procedures described in the methods section.
Supplementary Figure 5 shows the IR peak due to CH, NH and NH2 stretches, and the UV peaks on the same scale. The dispersion was the same as that used for the UV region and a logarithmic energy scale was needed to accommodate the energy range. The raw spectra are shown as Supplementary Figure 5a, the relative magnitudes are more apparent from the background subtracted spectra shown as Supplementary Figure 5b. The intensity of the IR peak has halved as the probe is moved to a position 17nm outside the specimen, while the intensity of the damage causing UV peaks has almost disappeared. Comparison of IR region spectrum taken using the ASU instrument (red, 15 meV FWHM zero loss) and the Rutgers instrument (black, 8 meV FWHM zero loss).
Supplementary Figure 6 shows a comparison between the spectrum acquired at ASU (15 meV FWHM) and the higher energy resolution spectrum acquired at Rutgers (8 meV FWHM). This graphically illustrates that the tail of the zero loss peak is the background in the IR region. Special efforts were taken in the design of the Rutgers spectrometer to improve the zero loss FWHM, and to reduce the tail of zero loss peak. These almost eliminate the background under the CH, NH and NH2 stretch peaks in the unsubtracted/raw spectrum. (a) Energy drift corrected spectrum showing the signal from the IR peaks on top of a background from the zero loss peak, and two separate background models used for the first peak and second peak, respectively (b) background-subtracted spectra.
Supplementary Figure 7 shows the background fitting procedure used to extract the peak intensities shown in Figs 2b, 2d, 4a and 4c. Separate power law fits (see Methods section) were used for the C=O peak and the composite peak from CH, NH and NH2 vibrations.