Enabling Liquid Scanning Electron Microscopy for Therapeutic Suspensions Using Vacuum-Compatible Liquid Capsules

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 <i>Z</i> 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. <i>Proc. Natl. Acad. Sci.</i> <b>2004</b>, <i>101</i>(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.