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Micropatterning of electron microscopy (EM) grids facilitates cryo-focused ion beam and cryo-electron tomography pipelines by optimally positioning cells and regions of interest in for milling and imaging (Engel, et al. 2019, Toro-Nahuelpan, et al. 2020, Engel and Vasquez, et al. 2021). Since EM grid micropatterning was introduced, we and other researchers have adopted new reagents for micropatterning EM grids using the Alveole PRIMO system which improve the efficiency of patterning and the stability of grid’s surface chemistry (Swistak et al. 2021, Sibert and Kim, et al. 2021). We have also introduced a sterilization step to make the process compatible with cell culture systems that do not contain antibiotics. Here, we present our protocol for efficient micropatterning of EM grids, which we have successfully used to micropattern grids for neuronal, cardiomyocyte, melanoma, and endothelial cell cultures. References: Engel L, Gaietta G, Dow LP, Swift MF, Pardon G, Volkmann N, Weis WI, Hanein D, Pruitt BL. Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies. J Micromech Microeng. 2019 Nov;29(11):115018. doi: 10.1088/1361-6439/ab419a. Epub 2019 Sep 26. PMID: 32879557. Toro-Nahuelpan M, Zagoriy I, Senger F, Blanchoin L, Théry M, Mahamid J. Tailoring cryo-electron microscopy grids by photo-micropatterning for in-cell structural studies. Nat Methods. 2020 Jan;17(1):50-54. doi: 10.1038/s41592-019-0630-5. Epub 2019 Nov 18. PMID: 31740821. Engel L, Vasquez CG, Montabana EA, Sow BM, Walkiewicz MP, Weis WI, Dunn AR. Lattice micropatterning for cryo-electron tomography studies of cell-cell contacts. J Struct Biol. 2021 Sep 11;213(4):107791. doi: 10.1016/j.jsb.2021.107791. PMID: 34520869. Léa Swistak, Anna Sartori-Rupp, Matthijn Vos, Jost Enninga. Micropatterning of cells on EM grids for efficient cryo-correlative light electron microscopy. Methods in Microbiology, 48, Elsevier, pp.95-110, 2021, doi: 10.1016/bs.mim.2020.11.001. Sibert BS, Kim JY, Yang JE, Wright ER. Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows. J Vis Exp. 2021 Sep 13;(175). doi: 10.3791/62992. PMID: 34570100. |
