Borum Raina, Reichert Paul, Li Zhao, Mohan Anne E, Bothe Jameson R, Chang Irene Yin-Ting, Su Yongchao, Zhang Yongqian
Analytical Research and Development, Merck & Co., Inc., Rahway, New Jersey 07065, United States.
Department of Protein and Structural Chemistry, Merck & Co., Inc., Rahway, New Jersey 07065, United States.
Anal Chem. 2025 Jul 15;97(27):14290-14300. doi: 10.1021/acs.analchem.5c01073. Epub 2025 Jul 3.
Liquid scanning electron microscopy (wet SEM) is an important tool that allows for high-resolution imaging of materials in their native liquid environments. Its application, however, to small organics and biologics that are often used in therapeutic suspensions has been hindered by challenges such as poor sample adhesion to the imaging window, high sensitivity toward radiation damage, and low contrast due to a low number. This work demonstrates strategies to overcome these limitations using a vacuum-compatible liquid cell, thereby expanding wet SEM's capabilities for in situ characterization of various therapeutic suspensions. We demonstrate that surface modification of the imaging window significantly enhances sample adhesion, an improvement previously reported to facilitate cell fixation (Thiberge et al. , (10), 3346-3351) but not tested for noncellular organic materials. Our direct comparison using vacuum-compatible liquid capsules with noncoated versus surface-coated imaging windows demonstrates the efficacy of this approach to fixate small molecules to enable wet SEM imaging. Imaging spacers enhance the bulk sampling efficiency for materials with heterogeneous particle sizes. Incorporating gadolinium acetate significantly improves contrast and resolution through surface adsorption, which increases the Z-contrast around the sample. Collectively, these approaches enabled a resolution of ∼10 nm for submicrometer protein particles in suspension. Elucidating the intricate particle attribute details across different solid-state forms in situ reveals how these structural attributes influence their rheological and pharmacokinetic behaviors. These findings demonstrate that the observed characteristics are highly dependent on the resolution of the results, proving their importance for advancing therapeutic process development and formulation design. Our study establishes the foundation for the versatility of wet SEM in exploring critical particle characteristics across a diverse range of organic and biological soft materials, enhancing its potential applications in drug development and formulation design. This work expands wet SEM's scope beyond conventional applications of cells and inorganic materials, positioning it as a powerful technique for in situ analysis of diverse organic and biological suspensions.
液体扫描电子显微镜（湿式扫描电子显微镜）是一种重要工具，可对处于天然液体环境中的材料进行高分辨率成像。然而，它在常用于治疗性混悬液的小分子有机物和生物制品方面的应用受到了诸多挑战的阻碍，如样品对成像窗口的附着力差、对辐射损伤高度敏感以及由于数量少导致对比度低等问题。这项工作展示了使用真空兼容液体池克服这些限制的策略，从而扩展了湿式扫描电子显微镜对各种治疗性混悬液进行原位表征的能力。我们证明成像窗口的表面改性显著增强了样品附着力，此前有报道称这种改进有助于细胞固定（蒂伯格等人，（10），3346 - 3351），但未对非细胞有机材料进行测试。我们使用具有未涂层与表面涂层成像窗口的真空兼容液体胶囊进行的直接比较证明了这种将小分子固定以实现湿式扫描电子显微镜成像方法的有效性。成像间隔物提高了具有不同粒径材料的整体采样效率。加入醋酸钆通过表面吸附显著提高了对比度和分辨率，这增加了样品周围的原子序数衬度。总体而言，这些方法使悬浮液中亚微米级蛋白质颗粒的分辨率达到了约10纳米。原位阐明不同固态形式下复杂的颗粒属性细节揭示了这些结构属性如何影响它们的流变学和药代动力学行为。这些发现表明观察到的特征高度依赖于结果的分辨率，证明了它们对推进治疗过程开发和制剂设计的重要性。我们的研究为湿式扫描电子显微镜在探索各种有机和生物软材料的关键颗粒特性方面的多功能性奠定了基础，增强了其在药物开发和制剂设计中的潜在应用。这项工作将湿式扫描电子显微镜的范围扩展到细胞和无机材料的传统应用之外，使其成为一种用于原位分析各种有机和生物混悬液的强大技术。
