A new method for vitrifying samples for cryoEM.

Almost every aspect of cryo electron microscopy (cryoEM) has been automated over the last few decades. One of the challenges that remains to be addressed is the robust and reliable preparation of vitrified specimens of suitable ice thickness. We present results from a new device for preparing vitrified samples. The successful use of the device is coupled to a new "self-blotting" grid that we have developed to provide a method for spreading a sample to a thin film without the use of externally applied filter paper. This new approach has the advantage of using small amounts of protein material, resulting in large areas of ice of a well defined thickness containing evenly distributed single particles. We believe that these methods will in the future result in a system for vitrifying grids that is completely automated.

[1]  Eugen Ermantraut,et al.  Perforated support foils with pre-defined hole size, shape and arrangement , 1998 .

[2]  Felix J. B. Bäuerlein,et al.  Micromachining tools and correlative approaches for cellular cryo-electron tomography. , 2010, Journal of structural biology.

[3]  W. Kühlbrandt The Resolution Revolution , 2014, Science.

[4]  Christopher Irving,et al.  Automation in single-particle electron microscopy connecting the pieces. , 2010, Methods in enzymology.

[5]  Roberto Marabini,et al.  Maximum-likelihood multi-reference refinement for electron microscopy images. , 2005, Journal of molecular biology.

[6]  Yifan Cheng Single-Particle Cryo-EM at Crystallographic Resolution , 2015, Cell.

[7]  N. Grigorieff,et al.  CTFFIND4: Fast and accurate defocus estimation from electron micrographs , 2015, bioRxiv.

[8]  Lei Jiang,et al.  Nanowire‐Haired Inorganic Membranes with Superhydrophilicity and Underwater Ultralow Adhesive Superoleophobicity for High‐Efficiency Oil/Water Separation , 2013, Advanced materials.

[9]  J. Dubochet,et al.  Cryo-electron microscopy of vitrified specimens , 1988, Quarterly Reviews of Biophysics.

[10]  Robert M Glaeser,et al.  Retrospective on the early development of cryoelectron microscopy of macromolecules and a prospective on opportunities for the future. , 2008, Journal of structural biology.

[11]  J M Carazo,et al.  A clustering approach to multireference alignment of single-particle projections in electron microscopy. , 2010, Journal of structural biology.

[12]  Christopher Irving,et al.  Appion: an integrated, database-driven pipeline to facilitate EM image processing. , 2009, Journal of structural biology.

[13]  B. Carragher,et al.  Spotiton: a prototype for an integrated inkjet dispense and vitrification system for cryo-TEM. , 2012, Journal of structural biology.

[14]  Prashant Rao,et al.  Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports , 2014, Scientific Reports.

[15]  C. Russo,et al.  Ultrastable gold substrates: Properties of a support for high-resolution electron cryomicroscopy of biological specimens , 2016, Journal of structural biology.

[16]  K. Adachi,et al.  A new method of preparation of a self-perforated micro plastic grid and its application. , 1965, Journal of electron microscopy.

[17]  Clinton S Potter,et al.  An Improved Holey Carbon Film for Cryo-Electron Microscopy , 2007, Microscopy and Microanalysis.

[18]  Anchi Cheng,et al.  Automated molecular microscopy: the new Leginon system. , 2005, Journal of structural biology.

[19]  A M Roseman,et al.  FindEM--a fast, efficient program for automatic selection of particles from electron micrographs. , 2004, Journal of structural biology.

[20]  J. Dubochet,et al.  Cryo-electron microscopy of viruses , 1984, Nature.