3/24/2024 0 Comments Types of tem grids![]() Recently, we observed that the same biological sample spotted onto similar grids coming from different batches led to dissimilar results. Finally, the air–water interface diffusion also needs to be taken into account ( Glaeser, 2018) as it also influences particle orientation and stability ( Noble et al., 2018 Jahagirdar et al., 2020). Sample buffer, sample concentration, temperature and humidity chamber, type of grids (Quantifoil, C-flat, and Holey), grid sample support (holey carbon, gold, nickel, and graphene), with or without an extra continuous carbon layer, glow discharge conditions, blotting time, and blotting force are parameters that are usually screened ( Passmore and Russo, 2016) to improve grid preparation. This task is not that straightforward, and many trials and errors are needed to obtain an optimal ice thickness ( Cho et al., 2013). To obtain high-resolution images, one needs first to vitrify a thin layer (ideally between 50 and 150 nm thickness) of a sample of interest. The methods used for each approach are described and the results obtained on a common specific case are reported.Įlectron microscopy of frozen hydrated samples (cryo-EM) is a powerful structural technique that allows the direct observation at a high resolution of functional macromolecular complexes in their near-physiological environment. ![]() This allowed us to diagnose the origin of grid preparation problems and to adjust glow discharge parameters. We report the use of several techniques to investigate the grids' characteristics, namely TEM, SEM, Auger spectroscopy and Infrared Interferometry. We recently run into such a case and finally found out that variations in the 3D reconstructions were systematically correlated with the grid batches that were used. Sample preparation on cryo-EM grids can give various results, from very thin ice and homogeneous particle distribution (ideal case) to unwanted behavior such as particles around the “holes” or complexes that do not entirely correspond to the one in solution (real life). 4Laboratoire de Physique de la Matière Condensée, Ecole Polytechnique, CNRS, IP Paris, Palaiseau, France.3Laboratoire de Physique des Interfaces et Couches Minces (LPICM), CNRS-UMR 7647, Ecole Polytechnique, IP Paris, Palaiseau, France.2Thales Research and Technology, Palaiseau, France.1Laboratoire de Biologie Structurale de la Cellule, BIOC, Ecole Polytechnique, CNRS-UMR7654, IP Paris, Palaiseau, France.Ramy Kazan 1 Gabrielle Bourgeois 1 Dominique Carisetti 2 Ileana Florea 3 Eric Larquet 4 Jean-Luc Maurice 3 Yves Mechulam 1 François Ozanam 4 Emmanuelle Schmitt 1 Pierre-Damien Coureux 1*
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