Gas-assisted annular microsprayer for sample preparation for time-resolved cryo-electron microscopy

Abstract

Time-resolved cryo electron microscopy (TRCEM) has emerged as a powerful technique for transient structural characterization of isolated biomacromolecular complexes in their native state within the time scale of seconds to milliseconds. For TRCEM sample preparation, microfluidic device [9] has been demonstrated to be a promising approach to facilitate TRCEM biological sample preparation. It is capable of achieving rapidly aqueous sample mixing, controlled reaction incubation, and sample deposition on electron microscopy (EM) grids for rapid freezing. One of the critical challenges is to transfer samples to cryo-EM grids from the microfluidic device. By using microspraying method, the generated droplet size needs to be controlled to facilitate the thin ice film formation on the grid surface for efficient data collection, while not too thin to be dried out before freezing, i.e., optimized mean droplet size needs to be achieved. In this work, we developed a novel monolithic three dimensional (3D) annular gas-assisted microfluidic sprayer using 3D MEMS (MicroElectroMechanical System) fabrication techniques. The microsprayer demonstrated dense and consistent microsprays with average droplet size between 6-9 μm, which fulfilled the above droplet size requirement for TRCEM sample preparation. With droplet density of around 12-18 per grid window (window size is 58×58 μm), and the data collectible thin ice region of >50% total wetted area, we collected ~800-1000 high quality CCD micrographs in a 6-8 hour period of continuous effort. This level of output is comparable to what were routinely achieved using cryo-grids prepared by conventional blotting and manual data collection. In this case, weeks of data collection process with the previous device [9] has shortened to a day or two. And hundreds of microliter of valuable sample consumption can be reduced to only a small fraction.

Two promising future developments of cryo-EM: capturing short-lived states and mapping a continuum of states of a macromolecule

“…First and foremost, increasing the data yield requires more coverage of thinly spread droplets on the grid before freezing. A promising new sprayer with 3D spraying geometry [46] proved to yield a greater number of smaller droplets than the current sprayer [25]. Continuing efforts on increasing the hydrophilicity of the grid surface (e.g.…”

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

“…The phenomenon that the ice is thicker on the trailing side than the leading side of the hole, with reference to the plunging direction, has not been observed previously(Chen et al, 2015; Lu et al, 2014; Lu et al, 2009). In some cases, the difference in ice thickness within one hole is as large as 200 Å.…”

“…Because of the presence of a mixing chamber inside the sprayer nozzle, the device is called an internal-mixing round-jet microsprayer (Lefebvre, 1980). Thus its design is different from that of Lu et al (Lu et al, 2014; Lu et al, 2009), which is characterized by external mixing, prefilming, spraying and discharge of a thin sheet of droplets from their sprayer (see also STAR Methods for our rationale for choosing the internal-mixing principle).…”

“…Recently developed time-resolved cryo-EM methods using the mixing-spraying method(Lu et al, 2009; Shaikh et al, 2014) are still limited in resolution as a consequence of low data yield compared to conventional cryo-EM using conventional pipetting/blotting. Our PDMS-based microsprayer can be readily integrated with other microfluidic components for time-resolved cryo-EM sample preparation, but this requires re-design of currently silicon-based chips(Lu et al, 2014; Lu et al, 2009) on a PDMS-based platform. In the future, the new microsprayer will be used as an integral component of a time-resolved microfluidic chip entirely based on PDMS.…”

“…In their experiment, a grid containing helical assemblies of the acetylcholine receptor was prepared in the standard way by pipetting-blotting and then sprayed with droplets of acetylcholine, setting off a reaction directly on the grid, which was subsequently plunged into the cryogen. In more recent implementations of time-resolved cryo-EM imaging, a reaction was performed directly in a microfluidic chip, and the reaction product was sprayed onto a dry EM grid(Lu et al, 2014; Lu et al, 2009; Shaikh et al, 2014). It is also worthwhile to mention that another kind of system called Spotion (Jain et al, 2012; Razinkov et al, 2016) was designed for dispensing specimen onto the EM-grid on the scale of picoliter to nanoliter volumes through piezo-electric inkjet technology, with which single droplets size is accurately controlled.…”