title: A Fast and Effective Microfluidic Spraying-Plunging Method for High-Resolution Single-Particle Cryo-EM.
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.
authors.affiliation: Department of Mechanical Engineering, Columbia University, New York, NY 10027, USA; School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, China.
authors.forename: Xiangsong
authors.initials: X
authors.lastname: Feng
authors.affiliation: Integrated Program in Cellular, Molecular, and Biophysical Studies, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA.
authors.forename: Ziao
authors.initials: Z
authors.lastname: Fu
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University New York, NY 10027, USA.
authors.forename: Sandip
authors.initials: S
authors.lastname: Kaledhonkar
authors.affiliation: Department of Mechanical Engineering, Columbia University, New York, NY 10027, USA.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Jia
authors.affiliation: Department of Biological Sciences, Barnard College, New York, NY 10027, USA.
authors.forename: Binita
authors.initials: B
authors.lastname: Shah
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University New York, NY 10027, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Jin
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University New York, NY 10027, USA.
authors.forename: Zheng
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Sun
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Chen
authors.affiliation: Department of Biochemistry and Molecular Biophysics, Columbia University New York, NY 10027, USA; Howard Hughes Medical Institute, Columbia University, New York, NY 10032, USA.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Grassucci
authors.affiliation: School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, China.
authors.forename: Yukun
authors.initials: Y
authors.lastname: Ren
authors.affiliation: School of Mechatronics Engineering, Harbin Institute of Technology, Harbin 150001, China.
authors.forename: Hongyuan
authors.initials: H
authors.lastname: Jiang
authors.affiliation: 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.
authors.forename: Joachim
authors.initials: J
authors.lastname: Frank
authors.affiliation: Department of Mechanical Engineering, Columbia University, New York, NY 10027, USA. Electronic address: qlin@columbia.edu.
authors.forename: Qiao
authors.initials: Q
authors.lastname: Lin
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods*; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D044085:Microfluidics / Q000295:instrumentation* / Q000379:methods
chemical_list: D004129:Dimethylpolysiloxanes; C013830:baysilon
keywords: EM grid preparation; PDMS-based microsprayer; apoferritin; ice thickness; time-resolved cryo-EM