Du Daniel X, Bartnik Adam C, Duncan Cameron J R, Choudhry Usama, Tabachnik Tanya, Sallah Chaim, Najafi Ebrahim, Yang Ding-Shyue, Maxson Jared M, Fitzpatrick Anthony W P
Cornell Laboratory for Accelerator-Based Sciences and Education, Cornell University, Ithaca, New York 14853, USA.
SLAC National Accelerator Laboratory, 2575 Sand Hill Rd, Menlo Park, California, 94025, USA.
Struct Dyn. 2025 Aug 26;12(4):044302. doi: 10.1063/4.0000770. eCollection 2025 Jul.
Cryo-electron tomography (cryo-ET) is a powerful modality for resolving cellular structures in their native state. While single-particle cryo-electron microscopy excels in determining protein structures purified from recombinant or endogenous sources due to an abundance of particles, weak contrast issues are accentuated in cryo-ET by low copy numbers in crowded cellular milieux. Continuous laser phase plates offer improved contrast in cryo-ET; however, their implementation demands exceptionally high-peak optical intensities. Instead, a novel experimental approach to enhance contrast in cryo-ET is to manipulate the phase of scattered pulsed electrons using ultrafast pulsed photons. Here, we outline the experimental design of a proof-of-concept electron microscope and demonstrate synchronization between electron packets and laser pulses. Furthermore, we show ultrabright photoemission of electrons from an alloy field emission tip using femtosecond ultraviolet pulses. These experiments pave the way toward exploring the utility of the ponderomotive effect using pulsed radiation to increase phase contrast in cryo-ET of subcellular protein complexes , thus advancing the field of cell biology.
冷冻电子断层扫描（cryo-ET）是一种用于解析天然状态下细胞结构的强大方法。虽然单颗粒冷冻电子显微镜在确定从重组或内源性来源纯化的蛋白质结构方面表现出色，这得益于大量的颗粒，但在冷冻电子断层扫描中，由于拥挤的细胞环境中拷贝数较低，弱对比度问题会更加突出。连续激光相位板可提高冷冻电子断层扫描中的对比度；然而，其实施需要极高的峰值光强度。相反，一种增强冷冻电子断层扫描对比度的新实验方法是使用超快脉冲光子来操纵散射脉冲电子的相位。在这里，我们概述了一个概念验证电子显微镜的实验设计，并展示了电子包与激光脉冲之间的同步。此外，我们展示了使用飞秒紫外脉冲从合金场发射尖端产生的超亮电子光发射。这些实验为探索利用脉冲辐射的有质动力效应来增加亚细胞蛋白质复合物冷冻电子断层扫描中的相位对比度铺平了道路，从而推动了细胞生物学领域的发展。
