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bioRxiv · 10.1101/2024.05.01.592094

Tuning ice thickness using the chameleon for high-quality cryoEM data collection

McGuire, K. L.·Cook, B. D.·Narehood, S. M.·Herzik, M. A.
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Abstract

Advances in single-particle cryogenic electron microscopy (cryoEM) now allow for routine structure determination of well-behaved biological specimens to high-resolution. Despite advances in the electron microscope, direct electron detectors, and data processing software, the preparation of high-quality grids with thin layers of vitreous ice containing the specimen of interest in random orientations remains a critical bottleneck for many projects. Although numerous efforts have been dedicated to overcoming hurdles frequently encountered during specimen vitrification using traditional blot-and-plunge specimen preparation techniques, the development of blot-free grid preparation devices provide a unique opportunity to carefully tune ice thickness, particle density, and specimen behavior during the vitrification process for improvements in image quality. Here, we describe critical steps of high-quality grid preparation using a SPT Labtech chameleon, evaluation of grid quality/ice thickness using the chameleon software, high-throughput imaging in the electron microscope, and recommend steps for troubleshooting grid preparation when standard parameters fail to yield suitable specimen. Video LinkContents of this manuscript are available as a video tutorial. This video can be found here

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McGuire, K. L., Cook, B. D., Narehood, S. M., Herzik, M. A.. 2024-05-04. Tuning ice thickness using the chameleon for high-quality cryoEM data collection. https://doi.org/10.1101/2024.05.01.592094

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