Sample deposition onto cryo-EM grids: from sprays to jets and back

Abstract

Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.

Keywords: cryo-EM; gas dynamic virtual nozzle; microfluidics; sample preparation; structure determination; time-resolved.

Figure 1

Characterization of droplet spreading after voltage-assisted spraying and freezing. (a) The average diameter of spread droplets on the grid is ∼200 µm, which is far in excess of the droplet size generated by the spray. The data are based on 50 observations in-flight and 48 droplets on three grids. (b) A large droplet which has not formed a thin film around the periphery of the drop. (c) Smaller spread droplets which produce areas suitable for imaging, highlighted with the blue arrows. The red dotted line shows the approximate droplet outline. The scale bar denotes 50 µm.

Figure 2

Deposition of droplets from Rayleigh jets. (a) Capillaries used for the generation of Rayleigh jets. (b) Frozen grids at low magnification in the electron microscope showing a clear strip but no droplet spreading. (c) The widths of the ice strips were approximately 200 and 60 µm for the 50 and 10 µm capillaries, respectively. All scale bars correspond to 100 µm.

Figure 3

GDVN used for cryo-EM grid preparation. (a) The GDVN device fitted within the current setup showing (I) the liquid inlet tubing, (II) the N₂ gas inlet tubing, (III) the position of the nozzle and (IV) the grid in the target position for spraying. (b) Microscopic image of the internal GDVN geometry used in this work with the sample and gas channels labelled. The scale bar denotes 100 µm. (c) Typical grids generated with the microfluidic GDVN device under three different conditions. On all three grids, liquid was deposited approximately as a stripe across the grid. The scale bar denotes 100 µm.

Figure 4

(a) High-speed imaging at the GDVN orifice of the jet/spray transition resulting from increasing gas and liquid flow rates. Bursts of 3000 consecutive frames were collected at 200 000 frames per second (fps) with an exposure time of 10 ns (pulsed laser illumination). The scale bar is 100 µm. (b) Droplet diameter distribution for a 220 SCCM N₂ flow. (c) Droplet-speed distribution at 220 SCCM.

Figure 5

(a) Low-magnification cryo-electron micrograph of a grid prepared using the microfluidic GDVN device in spraying mode (liquid at 4 µl s⁻¹, gas at 2 bar). The scale bar corresponds to 50 µm. (b) Representative high-magnification image of an area with thin ice used for data collection (the scale bar corresponds to 50 nm). (c) Single-particle reconstruction of apoferritin to 3.5 Å resolution with data collected from a single grid made using the GDVN nozzle under spraying conditions (liquid at 4 µl s⁻¹, gas at 2 bar).