We present a complex analysis and optimisation of dynamic conditions in the environmental scanning electron microscope (ESEM) to allow in-situ observation of extremely delicate wet bio-polymeric spherical particles in their native state. According to the results of gas flow and heat transfer simulations, we were able to develop an improved procedure leading to thermodynamic equilibrium between the sample and chamber environment. To quantify and hence minimise the extent of any sample deformation during specimen chamber pumping, a strength-stress analysis is used. Monte Carlo simulations of beam-gas, -water, and -sample interactions describe beam scattering, absorbed energy, interaction volume and the emission of signal electrons in the ESEM. Finally, we discuss sample damage as a result of drying and the production of beam-induced free radicals. Based on all experimental and simulation results we introduce a Delicate Sample Observation Strategy for the ESEM. We show how this strategy can be applied to the characterization of polyelectrolyte complex spherical particles containing immobilized recombinant cells E. coli overexpressing cyclohexanone monooxygenase, used as a model biocatalyst. We present the first native-state electron microscopy images of the viscous core of a halved polyelectrolyte complex capsule containing living cells.

中文翻译：

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基于仿真的 ESEM 热力学条件优化，用于球形聚电解质复合粒子在其原生状态下的动态原位研究

我们在环境扫描电子显微镜 (ESEM) 中对动态条件进行了复杂的分析和优化，以允许原位观察极其精细的湿生物聚合物球形颗粒的天然状态。根据气体流动和传热模拟的结果，我们能够开发一种改进的程序，导致样品和腔室环境之间的热力学平衡。为了量化样品室泵送过程中任何样品变形的程度并因此最小化，使用了强度-应力分析。束气、水和样品相互作用的蒙特卡罗模拟描述了 ESEM 中的束散射、吸收能量、相互作用体积和信号电子的发射。最后，我们讨论了由于干燥和束诱导自由基的产生而导致的样品损坏。基于所有实验和模拟结果，我们为 ESEM 引入了一种精细的样本观察策略。我们展示了这种策略如何应用于含有固定化重组细胞大肠杆菌的聚电解质复合球形颗粒的表征，大肠杆菌过表达环己酮单加氧酶，用作模型生物催化剂。我们展示了含有活细胞的对半聚电解质复合胶囊的粘性核心的第一张原生状态电子显微镜图像。