Miniaturized Sample Preparation for Transmission Electron Microscopy

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-andplunge-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.

Blotting-free and lossless cryo-electron microscopy grid preparation from nanoliter-sized protein samples and single-cell extracts

Henning Paul-Julius Stahlberg, Anastasia Syntychaki

We present a sample preparation method for cryo-electron microscopy (cryo-EM) that requires only 320 nL of sample to prepare a cryo-EM grid, depending on the protocol used. The sample is applied and spread on the grid by a microcapillary. The procedure does not involve any blotting steps, and realtime monitoring allows the water film thickness to be assessed and decreased to an optimum value prior to vitrification. We demonstrate that the method is suitable for high-resolution cryo-EM and will enable alternative electron microscopy approaches, such as single-cell visual proteomics. (C) 2016 The Author(s). Published by Elsevier Inc.

Elsevier BV2017

Etude par microscopie électronique de particules nanostructurées d'oxalate de cobalt précipitées en milieu aqueux

The understanding of material self-assembling and self-organization mechanisms, at mesoscale, is crucial for nanotechnologies development. Such structures, hierarchically organized in superlattices or colloïdal crystals, are observed in inorganic or organo-metallic precipitates. The classical characterization, using electron microscopy and X-ray diffraction, of a synthesized powder is inadequate to describe the hierarchically organized polycrystalline structure of the final particles. Here, we have highlighted the successive steps that lead to the spontaneous formation of a cobalt oxalate dihydrate (COD) colloïdal crystals that precipitate in aqueous media. The characterization of the final particles, made by X-ray and electronic diffraction, has allowed us to determine the controversial crystalline phase of the precipitated COD. The β and γ COD phases has been discriminated by comparing experimental results with simulations of X-ray diffractogram and electron diffraction patterns for these both structures. The precipitated COD corresponds to the γ phase indicating that it comes from solid dehydration of cobalt oxalate tetrahydrate. The final powder characterization, performed by X-ray diffraction (XRD), atomic force microscopy (AFM), low voltage high resolution scanning electron microscopy (LVHRSEM) and transmission electron microscopy (TEM), gives some diverging results about monocrystalline or polycrystalline nature of the precipitates. XRD and AFM analysis show a polycrystalline structure of particles whereas LVHRSEM, TEM and electron diffraction observations indicate a monocrystalline structure. This apparent contradiction has led us to elaborate a new characterization method allowing to follow the growth and the structural evolution of the precipitate as a function of time. The cryo- preparation techniques are well adapted as they allow freezing and direct observation of solid state suspension by electron microscopy. The freeze/drying method has been modified and used for both SEM and TEM, and has allowed us to study the morphological and structural evolution of the COD precipitate from nanoscale to mesoscale. In addition, TEM analysis of samples prepared by classical techniques has been performed to complement cryo-electron microscopy observations. The combination of these two methods show that COD precipitation is a complex multi-step process leading to the formation of a core/shell heterostructure. The anisotropic core is porous and partially crystalline. It is formed by agglomeration of isolated primary particles and agregated ones (15-20 nm sized secondary particles). The crystalline shell corresponds to the final particles faces and is made up by the layer by layer self-assembling and alignment of 5-7 nm sized crystalline primary particles. These nanoparticles are aligned and perfectly ordered in strings of nanograins in the layer structure. The self-assembling occurs in the last step of growth where we have a lower ionic strength and supersaturation inducing slower kinetics of growth and aggregation. Moreover, the crystalline order in the primary and secondary particles increase with the time of reaction consistent with a continuous process of primary particle nucleation and growth. Our results show that self-assembling occurs layer by layer with terraces, kinks and steps that are present on the faces. This reminds the layer by layer crystalline growth models but in this case, the buiding blocks are colloïdal instead of atomic or molecular. Based on these different results, we propose an original model describing the COD precipitate growth. In addition, we have studied the poly(methacrylic acid-sodium salt) effect on the COD precipitation. This additive (PMMA) appeared to be a very good growth inhibitor. The PMMA effect on the final particles morphology is dramatic. Its concentration variation in solution slows down the nanoparticles aggregation rates along a [101] preferential direction. The crystal habit can then be controlled and we have synthesized platelets, cubes and rods with tailored length. The use of cryogenic electron microscopy has been shown to be a powerful tool for the understanding of time dependent aspects of particle growth. The use of such techniques for the study of other systems, where the final precipitate appearance may hide particle substructures, will help to elucidate growth mechanisms and allow finer control of nanostructured materials for many applications. The complex self-assembling and self-organization processes could then be highlighted and studied on a nanometer to micrometer scale.

EPFL2004

Cryo-electron microscopy of membrane proteins

Henning Paul-Julius Stahlberg

Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.

Humana Press2013

Etude par microscopie électronique de particules nanostructurées d'oxalate de cobalt précipitées en milieu aqueux

EPFL2004