Miniaturizing EM Sample Preparation: Opportunities, Challenges, and “Visual Proteomics”

Abstract

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.

A microfluidic device for TEM sample preparation

“…Furthermore, we demonstrate a significantly lower liquid consumption with liquid volumes as small as in manual preparation protocols. 5,6 To demonstrate microfluidic consistency, we analyzed video recordings with respect to timing and duration of the microfluidic events on the five devices used with AAVs as the sample and NanoVan® as the stain. Fig.…”

“…This involves pipetting 3-5 μl of the sample liquid onto a TEM grid and letting it adsorb for around 10-60 s, depending on the specimen. 5,6 Then, the excess sample is manually blotted off the grid, using blotting paper. Immediately after blotting the sample, 3-5 μl of an aqueous stain solution is added to the grid.…”

“…Immediately after blotting the sample, 3-5 μl of an aqueous stain solution is added to the grid. 6 The excess stain is then blotted off, ideally leaving a uniform thin layer of stain liquid covering the adsorbed specimen. This thin film is left to dry, 7 embedding the specimen for TEM imaging and protecting it from dehydration.…”

“…However, they require special instrumentation and are significantly more complex and time-consuming than the manual preparation protocol. 6 Mukhitov et al proposed a microfluidic device for nsTEM grid preparation. 9 The TEM grid is confined in a microfluidic channel and the liquid handling for sample preparation is controlled by an external pressure pump.…”

Electron cryomicroscopy as a powerful tool in biomedical research

“…Apart from this, the emergence of new and the refinement of already established sample preparation methods has also greatly contributed to the resolution revolution, but its detailed description would exceed the scope of this review [21, 22]. …”