Abstract
Although microscopes and image-analysis software for electron cryomicroscopy (cryo-EM) have improved dramatically in recent years, specimen-preparation methods have lagged behind. Most strategies still rely on blotting microscope grids with paper to produce a thin film of solution suitable for vitrification. This approach loses more than 99.9% of the applied sample and requires several seconds, leading to problematic air-water interface interactions for macromolecules in the resulting thin film of solution and complicating time-resolved studies. Recently developed self-wicking EM grids allow the use of small volumes of sample, with nanowires on the grid bars removing excess solution to produce a thin film within tens of milliseconds from sample application to freezing. Here, a simple cryo-EM specimen-preparation device that uses components from an ultrasonic humidifier to transfer protein solution onto a self-wicking EM grid is presented. The device is controlled by a Raspberry Pi single-board computer and all components are either widely available or can be manufactured by online services, allowing the device to be constructed in laboratories that specialize in cryo-EM rather than instrument design. The simple open-source design permits the straightforward customization of the instrument for specialized experiments.
Highlights
During the 1980s, Jacques Dubochet and colleagues established that thin films of protein solutions can be vitrified and imaged by electron microscopy (Dubochet et al, 1988)
Dubochet’s method, which is still standard for cryo-EM, requires a few microlitres of protein solution to be applied onto an EM support grid coated with a carbon or gold film that contains numerous micrometre-scale holes
We present a simple and inexpensive cryo-EM specimen-preparation device where samples are applied to self-wicking EM grids as droplets generated from components adapted from a readily available ultrasonic humidifier
Summary
During the 1980s, Jacques Dubochet and colleagues established that thin films of protein solutions can be vitrified and imaged by electron microscopy (Dubochet et al, 1988). Sample loss has been addressed by using microcapillaries to apply small volumes of specimen solutions, with the cryoWriter device capable of applying less than 5 nl of sample to a conventional EM grid (Kemmerling et al, 2013; Arnold et al, 2016, 2017; Schmidli et al, 2019) This method has allowed the impressive determination of a structure of the human 20S proteasome to $3.5 Aresolution after isolation from 1 ml of cultured human cells (Schmidli et al, 2019). Owing to the use of a piezoelectric transducer to produce droplets of sample, and in homage to the Spotiton instrument that pioneered the use of self-wicking grids, we named our device Shake-it-off or SIO
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