A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM

Xiangsong Feng; Ziao Fu; Sandip Kaledhonkar; Yuan Jia; Binita Shah; Amy Jin; Zheng Liu; Ming Sun; Bo Chen; Robert A Grassucci; Yukun Ren; Hongyuan Jiang; Joachim Frank; Qiao Lin

doi:10.1016/j.str.2017.02.005

A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM

Structure. 2017 Apr 4;25(4):663-670.e3. doi: 10.1016/j.str.2017.02.005. Epub 2017 Mar 9.

Authors

Xiangsong Feng¹, Ziao Fu², Sandip Kaledhonkar³, Yuan Jia⁴, Binita Shah⁵, Amy Jin³, Zheng Liu³, Ming Sun⁶, Bo Chen⁶, Robert A Grassucci⁷, Yukun Ren⁸, Hongyuan Jiang⁸, Joachim Frank⁹, Qiao Lin¹⁰

Affiliations

¹ Department of Mechanical Engineering, Columbia University, New York, NY 10027, USA; School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, China.
² Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
³ Department of Biochemistry and Molecular Biophysics, Columbia University New York, NY 10027, USA.
⁴ Department of Mechanical Engineering, Columbia University, New York, NY 10027, USA.
⁵ Department of Biological Sciences, Barnard College, New York, NY 10027, USA.
⁶ Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
⁷ Department of Biochemistry and Molecular Biophysics, Columbia University New York, NY 10027, USA; Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA.
⁸ School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, China.
⁹ Department of Biochemistry and Molecular Biophysics, Columbia University New York, NY 10027, USA; Department of Biological Sciences, Columbia University, New York, NY 10027, USA; Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA. Electronic address: jf2192@cumc.columbia.edu.
¹⁰ Department of Mechanical Engineering, Columbia University, New York, NY 10027, USA. Electronic address: qlin@columbia.edu.

Abstract

We describe a spraying-plunging method for preparing cryoelectron microscopy (cryo-EM) grids with vitreous ice of controllable, highly consistent thickness using a microfluidic device. The new polydimethylsiloxane (PDMS)-based sprayer was tested with apoferritin. We demonstrate that the structure can be solved to high resolution with this method of sample preparation. Besides replacing the conventional pipetting-blotting-plunging method, one of many potential applications of the new sprayer is in time-resolved cryo-EM, as part of a PDMS-based microfluidic reaction channel to study short-lived intermediates on the timescale of 10-1,000 ms.

Keywords: EM grid preparation; PDMS-based microsprayer; apoferritin; ice thickness; time-resolved cryo-EM.

Published by Elsevier Ltd.

Publication types

Research Support, Non-U.S. Gov't
Research Support, N.I.H., Extramural

MeSH terms

Cryoelectron Microscopy / methods*
Dimethylpolysiloxanes / chemistry
Microfluidics / instrumentation*
Microfluidics / methods

Substances

Dimethylpolysiloxanes
baysilon

Abstract

Publication types

MeSH terms

Substances

Grants and funding