Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes

Abstract

Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.

Recent Technical Advances in Sample Preparation for Single-Particle Cryo-EM

“…Besides the classical techniques, new methods featuring creative strategies are emerging, such as those employing the HFBI grid (Fan et al, 2021) and the nanofluidic chip (Huber et al, 2022). In these new methods, special materials or unique designs are used to seek breakthroughs against the bottleneck of sample preparation, which is greatly encouraging for the cryo-EM community.…”

“…A subsequent study further showed that a modular microfluidic system, featuring a dwell time as low as 16 ms and a minimal solution volume of ~20 μl, improved the particle distribution of apoferritin and eliminated the aggregation of the CSN 5H138A -SCF-N8 Skp2/Cks1 complex in the grid hole (Mäeots et al, 2020). Recently, a nanofluidic chip has been reported in preparing the cryo-EM samples with increased reproducibility (Huber et al, 2022). The cryo-chip contains several nanofluidic channels formed by thin silicon-rich nitride (SiN x ) membranes to provide a predefined space to control ice thickness while maintaining electron transparency for cryo-EM imaging (Figure 2B).…”

“…Consequently, generating high-quality ice for data collection is time consuming (Klebl et al, 2020). Furthermore, although the nanofluidic cryo-chip featuring the SiN x membrane eliminates the AWI problem, the solid-liquid interfaces can also affect the distribution of particles (Huber et al, 2022).…”

“…Notably, the design of the nanofluidic system also represents an attempt to automate the cryo-EM workflow and facilitate the sample preparation process (Huber et al, 2022). Whereas automation of the cryo-EM data processing has been widely studied (Li et al, 2020;Maruthi et al, 2020;Stabrin et al, 2020), research on automation of the sample preparation process remains limited.…”

Perspective: Biochemical and Physical Constraints Associated With Preparing Thin Specimens for Single-Particle Cryo-EM

“…Thus, as is summarized in Table 1, adsorption onto solid surfaces such as glow-discharge treated carbon film or graphene oxide may produce favorable results for some proteins, while adsorption of other proteins to the same substrates results in preferred orientation or even severe particle damage. It therefore seems likely that the same, specimen-dependent outcome will prove to be true for adsorption of proteins onto the silicon nitride windows of microfluidic EM grids (Huber et al, 2022).…”