Miniaturized Sample Preparation for Transmission Electron Microscopy

Abstract

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for "visual proteomics," or "lossless" total sample preparation for quantitative analysis of complex samples.

Challenges and opportunities in cryo-EM single-particle analysis

“…As the tools become more developed, and the downstream protocols become more automated, we will want to examine more challenging samples, including those that may be less abundant, that contain transiently interacting factors, and that generally represent highly dynamic and heterogeneous assemblies. It should be possible to perform relatively crude purifications, even starting from cell lysate (53,54), and to capture a spectrum of heterogeneity within the imaged sample (55). Accomplishing such a task for higher-resolution studies would feed off of developments in all the downstream steps, especially as pertains to specimen vitrification (which can disrupt macromolecular integrity through destructive forces at the air-water interface (56)), data collection (which will benefit from further speedups (57, 58)), and image analysis (which can become complex for highly heterogeneous cases).…”

“…In practice, it is not clear how many impurities it is possible to tolerate. Nonetheless, such "lysate-to-structure" methods represent the first steps toward the cryo-EM version of structural (or visual) proteomics (53,54), and with the right approach (and perhaps mild biochemical enrichment), one can envision the possibility of taking relatively crude material and determining structures of many, or at least some, core macromolecules or macromolecular assemblies.…”

A microfluidic device for TEM sample preparation

“…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.…”

“…Alternative methods to obtain a consistent nsTEM sample preparation employ contact pin-printing techniques where pipetting robots automatically dispense liquids onto the TEM grid. 5,[10][11][12][13] These approaches have some advantages over the manual preparation, such as reduction of liquid volumes and the possibility for automation. However, they require special instrumentation and are significantly more complex and time-consuming than the manual preparation protocol.…”

The His-tag as a decoy modulating preferred orientation in cryoEM

“…These supports may also be chemically modified to promote specific, high-affinity interactions with particles (Llaguno et al, 2014, Kelly et al, 2010a, Kelly et al, 2008, Kelly et al, 2010b, Han et al, 2012, Benjamin, Wright, Bolton, et al, 2016, Crucifix et al, 2004, Wang, Liu, et al, 2020, Yeates et al, 2020. Finally, new, fast approaches for depositing samples on a grid have been developed (Arnold et al, 2017, Schmidli et al, 2018, Dandey et al, 2020, Ravelli et al, 2019. One of the strategies for changing interactions of macromolecules with a support, and with the water-air interface, is to chemically modify the molecule itself without changing the chemical properties of the support.…”