Nanofluidic chips for reproducible cryo-EM sample preparation with picoliter sample volumes

Introduction

Cryogenic transmission electron microscopy (cryo-EM) is a technique for high-resolution imaging of radiation-sensitive biological macromolecules under near-native conditions.

Sample Preparation Techniques

Cryo-EM sample preparation techniques rely on rapid cooling of an aqueous suspension of biological macromolecules to obtain a thin layer of amorphous ice containing the solute.

Challenges in Current Implementations

Problems associated with current implementations are considered a major bottleneck to realizing the full potential of cryo-EM:

• Excessive sample consumption
• Difficulty in reproducibly obtaining ice conditions suitable for high-resolution imaging
• Extensive contact of solute molecules with a large air-water interface

These issues result in only a small fraction of the imaged molecules contributing useful information for the final 3D reconstruction.

Time Resolution Limitations

Poor time resolution of these methods furthermore precludes the visualization of dynamic structural changes that provide critical biological insight.

Proposed Solution

In our laboratory, we have developed technology to design nanofluidic MEMS devices suitable for cryo-EM imaging. We propose to explore product development of a nanofluidic chip for reproducible sample preparation with picoliter volumes.

Nanofluidic Chip Design

The sample is contained in nanochannels formed between membranes of an electron-transparent material, thereby controlling ice thickness and avoiding the formation of an air-water interface.

Advantages of the New Method

This method has all features to push cryo-EM to new frontiers:

1. Requires minute amounts of sample
2. Robustly provides uniform and customizable thickness gradients across the sampling area
3. Time resolution that is limited only by the vitrification process itself

Future Opportunities

Our CryoChip will provide entirely new opportunities for time-resolved cryo-EM imaging and high-throughput applications in structure-based drug design for the pharmaceutical industry.

Collaboration with Industry

We will liaise with an industrial partner experienced in MEMS probe fabrication to explore process optimization and the viability of larger-scale production.