cryoWriter: a blotting free cryo-EM preparation system with a climate jet and cover-slip injector

Abstract

Electron microscopy (EM) introduced a fast and lasting change to structural and cellular biology. However, the sample preparation is still the bottleneck in the cryogenic electron microscopy (cryo-EM) workflow. Classical specimen preparation methods employ a harsh paper-blotting step, and the protein particles are exposed to a damaging air-water interface. Therefore, improved preparation strategies are urgently needed. Here, we present an amended microfluidic sample preparation method, which entirely avoids paper blotting and allows the passivation of the air-water interface during the preparation process. First, a climate jet excludes oxygen from the sample environment and controls the preparation temperature by varying the relative humidity of the grid environment. Second, the integrated "coverslip injector" allows the modulation of the air-water interface of the thin sample layer with effector molecules. We will briefly discuss the climate jet's effect on the stability and dynamics of the sample thin films. Furthermore, we will address the coverslip injector and demonstrate significant improvement in the sample quality.

Fig. 1. Cryo-EM sample preparation using the cryoWriter system with a climate jet and “coverslip”-injector. (A) A cryo-EM grid (1), held by a temperature-controlled tweezer (2), is placed on a “dew-point” stage (3) with good thermal contact. The climate jet (4) allows control of the temperature and the relative humidity around the grid. Note the groove (5), which allows the conditioned gas to stream on both sides of the grid (Fig. 2D). Next, the grid is primed with a thin layer of the sample by a microcapillary (6). (B) During the sample priming, a quick spray of surface-active effector molecules by a nebulizer coats the AWI. (C) After a short settling time (≈360 ms), the grid is withdrawn by temperature-controlled tweezers (2), flipped by 90° into a vertical position, and plunged in a bath of liquid ethane (7).

Fig. 2. Climate jet and coverslip injector. (A) Overview. The climate jet system controls the temperature and humidity of a N₂-stream. After passing through a pressure reducer (1), the stream is split into two lines, which are individually controlled by mass-flow controllers (2). One line is humidified by a commercial humidifying column (3). Next, the two streams are combined and enter a temperature conditioner (4), which adjusts the temperature of the two combined N₂-streams. Finally, the gas stream enters the nozzle (5), forming a jet stream flowing over the cryo-EM grid. Note that the temperature and relative humidity are also measured in the nozzle, and two PID controls regulate the two mass-flow controllers and the temperature conditioner. (B) Details of the temperature conditioner. A water-cooled (6) Peltier element (7) efficiently heats or cools the gas stream, which flows between a metal grill (8) and plastic cap (9) for efficient heat transfer between the gas stream and the Peltier element. (C) Arrangement of the climate jet on the dew-point stage (see also Fig. 1). The nozzle (5) points toward the cryo-EM grid (10), placed with good thermal contact on top of an indentation in the temperature-controlled dew-point stage (11). (D) Details of the climate jet outlet nozzle, the N₂-flowfield, and the nebulizer: the conditioned N₂-stream enters a chamber (12), also hosting the sensors for the humidity and temperature measurement. A microcapillary (13), filled with a solution of effector molecules, points to the middle of a narrow restriction (14), where the stream velocity is at its maximum. The microcapillary is connected to a high-precision pump system to inject a small amount of effector molecules into the gas stream, which is then transported towards the cryo-EM grid (10) placed above an indentation in the dew-point stage (11).

Fig. 3. Comparison of the preparation of a membrane protein with a classical preparation system (A) and the same protein using the cryoWriter setup (B). Representative class averages are shown.

Fig. 4. Effect of the coverslip injector with plain water (A and B) or Chaperonin 60 (C and D) as the sample. The grids were prepared without injection (A and C) or active spray (B and D). A pulse of 600 ms of an OG solution was applied simultaneously with reaspiration of the sample. See also Fig. 1 and ESI Fig. 2. Scale bars: 200 nm.