A cold field emission gun optimized for cryo-EM applications.

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Poster Session 4

Electron microscopy, CryoEM, cold field emission gun, Single particle analysis 

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A cold field emission gun (FEG) is characterized by its high brightness and the low energy spread of the electron beam. The cold FEG can further be optimized for high brightness applications by increasing the extraction voltage. The higher brightness will improve the signal to noise ratio of applications like high-resolution scanning transmission electron microscopy (HR-STEM) and energy dispersive x-ray spectroscopy mapping. On the other hand, some applications favor a further lowering of the energy spread which can be achieved by lowering the extraction voltage. One example is electron energy loss spectroscopy (EELS), where the lower energy spread will increase the resolution of the energy spectrum. Another example is high-resolution transmission electron microscopy (HR-TEM) where the lower energy spread leads to better contrast at high frequencies by lowering the impact of the chromatic aberration which will result in a higher spatial resolution of the 2D image. The flexibility to tune the microscope for high brightness or low energy spread is advantageous for microscopes used for material science applications, where there is a need to characterize the specimen with a variety of (analytical) imaging techniques. This flexibility is much less required in life science applications like cryo-EM single particle analysis (SPA) and tomography that are mostly performed in HR-TEM mode. The stability requirements are however more stringent for cryo-electron microscopes since especially SPA requires fully automated data acquisition runs which can take from several hours to several days. 

To further improve 3D resolution of proteins and protein complexes obtained by cryoEM, the Thermo Fisher high-end cryo electron microscope (Krios) can now be configured with a 300 kV CFEG which is optimized for life science applications (E-CFEG). We will show how the lower energy spread leads to high contrast and therefore high 2D and 3D spatial resolution in SPA, and why the lower brightness setting of the E-CFEG is not negatively impacting these resolutions. One characteristic of a cold field emitter is that the beam current stability is directly related to the vacuum condition of the field emitter tip which slowly decays over the course of hours. After the beam current drops below a certain level, the tip must be regenerated via a short heating of the tip. This procedure is called tip flashing and takes less than a minute and must be repeated during a long SPA data run. The flashing is completely automated in data acquisition programs like Tomo and EPU and triggered based on the monitored level of the beam current.