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Abstract：
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.
摘要：
通过微晶电子衍射（MicroED）研究了样品厚度与获得的数据质量之间的关系。制备了几个电子显微镜（EM）网格，其中包含来自同一结晶批次的相似尺寸的蛋白酶K微晶。将每个网格转移到聚焦离子束和扫描电子显微镜中，然后将晶体系统地减薄为95至1,650nm厚的薄片。MicroED数据在120-，200-,或300kV加速电压。薄片厚度以相应的非弹性平均自由程的倍数表示，以比较不同加速电压的结果。使用标准晶体学测量来评估数据的质量和随后确定的结构。从晶体薄片中以相似的质量可靠地确定了结构，最高为非弹性平均自由程的两倍。对于所有三个加速电压，在平均自由程的三倍处观察到较低分辨率的衍射。但是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，并对所有低温EM方法都有影响。
