Cryo transmission electron microscopy (TEM) provides structural information of a specimen close to its natural state without any disturbance, due to the specimen preparation process, which exclude chemical reactions and physical stimulations. Recently, cryo TEM produces very exciting results of structural biology in combination with single particle analysis and electron tomography, since application field of the method expand to non crystalline samples or huge molecules.

We developed a new generation cryo TEM, which achieves high throughput and high usability. This microscope equips 200 kV field emission gun (FEG). Users can choose it from a Schottky-type (TFEG) or a cold FEG (CFEG). Since the energy spread of the emitted electrons from the CFEG is about 50% of TFEG and the size of the virtual source is less than 10 nm, the electron beam has a high coherences. With such beam, cryo TEM image has high contrast due to its high spatial coherence and is less affected by chromatic aberration due to its high temporal coherence. In low dose imaging, where the image resolution is mainly determined with dose density for the image. In the low dose density, S/N of image mostly determined by a statistical noise of electrons, since dose density in cryo TEM is typically several tens of electrons for angstrome square. Namely, the resolution is determined with the competition between the statiscal noise and image contrast. It means CFEG has posibility to have higher resolution for cryo TEM works.

Since this microscope also has dedicated cryo stage, cryo TEM observation can be performed at low temperature < 100 K  and with low grow ratio of ice contamination. In addition, this cryo stage is compatible for multi-specimen auto-loader, so users can exchange specimens automatically. It also has some automation functions, such as liquid nitrogen auto-refill system and auto acquisition software (JEOL Automated Data Acquisition System: JADAS). These automation functions will help users to perform high throughput works. On another front, the electron gun chamber and the TEM column are evacuated with sputter ion pump and turbo-molecular pump, because of this, a sample is kept in oil-free environment.

In addition, this microscope is compatible with omega-type energy filter and Zernike or hole-free phase plate. The cryo specimens exhibit low contrast in TEM images even using large defocus phase contrast imaging. The Cryo TEM is more advantageous when it is combined with these techniques of contrast enhancement.

Figures:

Fig. 1 The appearance of the new cryo TEM. Multi-specimen auto-loader exists inside green box (Red arrow).

Fig. 2 An ice embedded GroEL image taken by the new cryo TEM. Observation was done at 200 kV and recorded onto 1k x 1k CCD. This image was taken with a slight defocus (about -3 μm).

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EMC Abstracts - https://emc-proceedings.com/abstract/development-of-new-generation-cryo-tem/