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Sample thickness is a known key parameter in cryo-electron microscopy (cryo-EM) and can affect the amount of high-resolution information retained in the image. Yet, common data-acquisition approaches in single-particle cryo-EM do not take it into account. Here, it is demonstrated how the sample thickness can be determined before data acquisition, allowing the identification of optimal regions and the restriction of automated data collection to images with preserved high-resolution details. This quality-over-quantity approach almost enti

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