Coating-Free Low-Voltage Scanning Electron Microscopy for Native-Surface Imaging across Scale, Depth, and Time

Conspectus Organic and hybrid materials derive their functions not only from molecular structure but also from how molecules organize across a hierarchy of length scales─their meso-hierarchies. Understanding such materials therefore requires one to observe them in their native states before imaging alters their structures. Scanning electron microscopy (SEM) is one of the most direct pathways to visualize these hierarchies; however, imaging organic materials has long been hindered by an intrinsic dilemma: high-resolution imaging relies on electrons, which inevitably causes charging on insulating specimens. While conductive metal coatings represent routine solutions, they inevitably alter the specimens by adding mass, obscuring nanostructural details, complicating elemental analysis, and preventing repeated observations of treated specimens. We encountered an analogous problem in transmission electron microscopy (TEM), where electron irradiation causes ionization that can alter the composition of insulating organic molecules. Our solution was to encapsulate individual molecules within carbon nanotubes or mount them onto their conductive walls to dissipate charge and suppress ionization, forming the foundation of a field we have termed “cinematic chemistry”. Fifteen years ago, we investigated whether an analogous strategy could be applied to observe the native surface instead of its metal-coated replica using SEM. The results revealed that observing the native surface was far more complex than merely removing the coating; it required simultaneously optimizing electron optics, landing voltage, and specimen preparation, alongside utilizing a substrate made of a conductive material, such as indium tin oxide (ITO), to preserve the native morphology of dispersed organic assemblies. To demonstrate this optimization, we spin-coated self-assembled [60]fullerene vesicles and target proteins onto precleaned ITO substrates. The uncoated specimens were then imaged at landing voltages ranging from several hundred volts to a few kilovolts. Consequently, individual avidin molecules and single gold nanoparticles on the vesicles were directly resolved on the native surface, entirely avoiding the granularity introduced by metal coatings. Over the years that followed, we realized that coating-free SEM should not be the ultimate goal. Rather, preserving the native surface allows a single instrument to continuously observe the same specimen across three dimensions: scale, depth, and time. We traced the chitin scale of a deepsea snail from its millimeter-sized fracture surface down to its constituent nanofibers; we observed the buried layers of a polymer composite and a solar cell cathode simply by raising the landing voltage. We followed the same specimen through a chemically converted perovskite precursor and the reversible, beam-driven folding of a free-standing fullerene nanofilm. The results collectively revealed the meso-hierarchy that links the molecular structures of self-assembled organic architectures and organic–inorganic hybrids to their macroscopic functions. This Account summarizes what 15 years of such imaging have taught us: to identify substrates and cleaning protocols that preserve a native surface, how to select the optimal landing voltage for measuring the density and depth of interest, and what limitations define the chosen approach. These developments suggest a broad philosophy for observing materials in their native chemical states.