4. Discussion

We have developed a system for fully automated acquisition of 1000 electron micrographs a day. The Leginon system can be operated continuously over a 24 hour period and the only human intervention necessary is to fill the liquid nitrogen dewar.

There are significant variations in the percentage of acceptable images for a given grid and we have used grids of similar quality in all of the results discussed. Using very simple modeling criteria for feature selection, the number of acceptable images acquired by the Leginon system was comparable to the numbers acquired by a human operator. We have thus shown that we have developed a fully automated system that performs at least as well as a student microscopist.

There are a number of techniques that could be used to further improve the automated system's performance. First, the quality assessment could be improved by using indexing methods to more accurately identify diffraction spots and eliminate spurious spots. Other criteria (e.g. Friedel's law) could also be used to assess the quality of the crystals. Second, the feature identification and targeting methods could be much improved. For example, the variance of the intensities of the identified features is an indicator of the uniformity of the crystal thickness which should be incorporated into the targeting decision. Third additional targeting schemes should be used. For example, within large uniform crystals multiple targets should be identified. We intend to incorporate these improvements into the next application of the Leginon system which will be focused on automated acquisition of cryo-electron micrographs.

Page 5 of 7