Automated Acquisition of Cryo Electron Micrographs Using Leginon

Molecular microscopy is one of the most important structural approaches in cell biological investigations and can provide insight into complex biological questions that no other technique can provide. Currently, the technique typically requires the acquisition of very large numbers of transmission electron micrographs from frozen hydrated specimens using low dose techniques. The field is constrained by manual data acquisition methods that are slow, labor-intensive and result in a very low percentage of suitable images. We have developed a system, called Leginon, for automatically acquiring images from a transmission electron microscope^1,2. Our first prototype of this system demonstrated that we could acquire 1000 high magnification images per day from negatively stained catalase crystals. We have now extended this system to acquire low dose images of specimens embedded in vitreous ice.

Methods

Specimens were prepared on Quantifoil grids³ using techniques which have been described previously⁴. The Leginon system uses a Philips CM200 TEM and a Gatan MSC CCD camera and is controlled by the emScope software library⁵. The overall acquisition protocol requires (i) obtaining a low magnification image [660x] of a grid square from a Quantifoil grid (fig. 1a); (ii) automatically identifying holes containing ice of suitable thickness; (iii) acquiring an intermediate magnification image [6600x] of the identified hole (fig. 1b); (iv) identifying features of interest within the hole; (v) focusing at high magnification [38,000x] and finally (vi) acquiring a high magnification image (fig 1c,d).

Figure 1. (a) TMV embedded in vitreous ice suspended over holes in a Quantifoil grid [660x]. (b) Identified hole indicated by a cross in (a) imaged at 6600x. (c) Center of hole from (b) imaged at 38,000x; individual TMV filaments can be identified. (d) Similar procedures resulting in images of microtubules at 38,000x.

a	b
c	d

In step (ii) holes in the Quanitfoil grid are automatically identified using a cross correlation template matching and thresholding procedure combined with a filter to identify the geometric parameters of the Quantifoil lattice. The thickness of the ice for a given hole is estimated⁶ and a threshold on the ice thickness is set to identify holes for further analysis in step (iii). Efficient implementation of the system required improvements in the positioning accuracy of the goniometer which were achieved using modeling techniques described elsewhere⁷.

Results

We have tested the Leginon system using specimens of TMV and microtubules embedded in vitreous ice. The system can acquire over 1000 high magnification images a day. We are currently assessing the performance of the system and comparing it against the performance of a human operator.

References

1. C.S. Potter et al., Proc. Microscopy and Microanalysis (1998) 8-9.
2. C. S. Potter et al., Submitted for publication.
3. E. Ermantraut, K. Wohlfart and W. Tichelaar, Ultramicroscopy, 74 (1998) 75-81.
4. J. Dubochet et al., Quart Rev Biophys, 21 (1988) 129-228.
5. N. Kisseberth et al., J. Struct. Biology, 120 (1997) 309-319.
6. R. Eusemann et al., J. of Microscopy, 128 (1982) 239-249.
7. J. Pulokas et al., Submitted to this conference.
8. Support is provided by the NSF (9730056) and the IBM Shared University Research program.

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