Automated vitrification of cryo-EM samples with controllable sample thickness using suction and real-time optical inspection

Abstract

The speed and efficiency of data collection and image processing in cryo-electron microscopy have increased over the last decade. However, cryo specimen preparation techniques have lagged and faster, more reproducible specimen preparation devices are needed. Here, we present a vitrification device with highly automated sample handling, requiring only limited user interaction. Moreover, the device allows inspection of thin films using light microscopy, since the excess liquid is removed through suction by tubes, not blotting paper. In combination with dew-point control, this enables thin film preparation in a controlled and reproducible manner. The advantage is that the quality of the prepared cryo specimen is characterized before electron microscopy data acquisition. The practicality and performance of the device are illustrated with experimental results obtained by vitrification of protein suspensions, lipid vesicles, bacterial and human cells, followed by imaged using single particle analysis, cryo-electron tomography, and cryo correlated light and electron microscopy.

Electron microscopy analysis of soft materials with freeze‐fracture techniques

“…Liquid nitrogen, which is used for typical rapid cooling, has a low heat capacity, which can cause the Leidenfrost effect that prevents the specimen from contacting liquid nitrogen, making it difficult to obtain a sufficient cooling rate of the samples 49 . For this purpose, mainly liquid ethane or propane is used 20,50,51 . As an optimized procedure, the sample is immersed in low‐temperature liquid ethane or propane container surrounded by liquid nitrogen for stable, high‐speed rapid freezing (Figure 1b).…”

“…[10][11][12][13][14][15][16] Depending on the size and properties of the material, sophisticated pretreatment, and observation techniques are essentially required. Scanning electron microscopy (SEM), 2,17,18 transmission electron microscopy (TEM), 11,[19][20][21][22] atomic force microscopy (AFM), [23][24][25] and x-ray diffraction (XRD) [26][27][28] are generally used to analyze structures that have nanometer scales. SEM and TEM, frequently used for the visualization of nano-micromaterial structures, use the interaction of materials with high-energy electrons with wavelengths smaller than light to discriminate the structure.…”

Maintaining the momentum in cryoEM for biological discovery

“…Both the specimen preparation and microscope hardware can influence the time taken and success of the screening step. Newer specimen preparation technologies provide a view of the grid from the freezing process which can be used to judge the quality of the grid and ice without loading into a microscope [15,[27][28][29][30]. Screening for appropriate ice thickness, and particle concentration and distribution can be done manually, however this is dependent on microscope operator experience, and is always a subjective judgement.…”

Vitrocam: A simple low cost Vitrobot camera for assessing grid quality

“…At the very least, it is possible to tell when a grid did not successfully wick and that it will have ice that is too thick and thus should be discarded. Recently, a novel sample preparation device coupled to a Linkam stage 11 was developed that utilizes a light microscope to monitor the liquid aspiration process and determine the right moment for vitrification, so to improve control and reproducibility of the resulting ice thicknesses. All these devices have thus incorporated cameras into the grid preparation process to improve success rates and limit wasting time and money on grids that are not suitable for imaging.…”