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

Area of Science:

• Structural Biology
• Biophysics
• Microfluidics

Background:

• Cryoelectron microscopy (cryo-EM) requires high-quality samples with uniform vitreous ice.
• Conventional sample preparation methods like pipetting-blotting-plunging can be inconsistent.
• Developing automated and reproducible sample preparation is crucial for advancing cryo-EM.

Purpose of the Study:

• To introduce a novel spraying-plunging method for cryo-EM grid preparation.
• To achieve controllable and highly consistent vitreous ice thickness.
• To demonstrate the utility of this method for high-resolution structure determination and time-resolved cryo-EM.

Main Methods:

• Development of a polydimethylsiloxane (PDMS)-based microfluidic sprayer.
• Application of the spraying-plunging technique to prepare cryo-EM grids.
• Testing the method with apoferritin for structural analysis.

Main Results:

• The microfluidic sprayer successfully produced cryo-EM grids with consistent vitreous ice thickness.
• High-resolution structural determination of apoferritin was achieved using the prepared grids.
• The method shows potential for replacing conventional sample preparation techniques.

Conclusions:

• The spraying-plunging method offers a reproducible way to prepare cryo-EM samples.
• This technique is suitable for routine high-resolution cryo-EM studies.
• The microfluidic sprayer is a promising tool for time-resolved cryo-EM, enabling the study of transient biological intermediates.