The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.

通过微晶电子衍射 (MicroED) 研究样品厚度与所获得数据质量之间的关系。制备了几个含有来自同一结晶批次的相似尺寸的蛋白酶 K 微晶的电子显微镜 (EM) 网格。每个网格被转移到聚焦离子束和扫描电子显微镜中，然后晶体被系统地减薄成厚度在 95 到 1,650 nm 之间的薄片。MicroED 数据是在 120、200 或 300 kV 加速电压下收集的。薄片厚度以相应非弹性平均自由程的倍数表示，以允许比较不同加速电压的结果。使用标准晶体学测量评估数据的质量和随后确定的结构。从结晶薄片到非弹性平均自由程的两倍，可以可靠地确定具有相似质量的结构。在所有三种加速电压的平均自由程的三倍处观察到较低分辨率的衍射，但数据质量不足以产生结构。最后，从厚度超过计算的非弹性平均自由程的四倍的薄片中没有观察到相干衍射。这项研究对理想的样品厚度进行了基准测试，并对所有冷冻电镜方法都有影响。从厚度超过计算的非弹性平均自由程的四倍的薄片中没有观察到相干衍射。这项研究对理想的样品厚度进行了基准测试，并对所有冷冻电镜方法都有影响。从厚度超过计算的非弹性平均自由程的四倍的薄片中没有观察到相干衍射。这项研究对理想的样品厚度进行了基准测试，并对所有冷冻电镜方法都有影响。