Three-dimensional electron diffraction (3DED) has become a powerful method for structure determination of nano-/micron-sized crystalline materials. Due to their negative charge, electrons interact with both atomic nuclei and surrounding electron clouds in matter. While this strong interaction enables the study of nano-/micron-sized crystals, it also induces electron-beam damage during 3DED experiments. When electrons interact with a crystal, they can cause atomic displacement and bond breakage, which affects the structure and/or the chemistry of the specimen. This damage poses a significant challenge for 3DED studies of beam-sensitive materials.

In this thesis, eight beam-sensitive crystalline materials were investigated using 3DED, including pharmaceuticals, zeolites, and metal-organic frameworks (MOFs). To address electron-beam damage, several data acquisition strategies were developed to preserve beam-sensitive materials during 3DED experiments. These strategies include: 1) using low electron fluence, 2) cryo-cooling, and 3) the low-dose cryo-cRED (cryogenic continuous rotation electron diffraction) method, which combines the former two. These strategies reduce electron-beam damage and enhance the reliability of 3DED.

Additionally, a glovebox-assisted sample preparation workflow was developed to prepare cryo-samples under a controlled atmosphere. This approach enables 3DED studies of beam-sensitive materials that are also sensitive to air.

Using 3DED at room temperature, two new piroxicam (PXM) polymorphs were identified. To mitigate electron-beam damage, the cumulative electron fluence per dataset was reduced by adjusting data acquisition parameters related to electron flux and recording time. The structure information provided insight into the structure–property relationship between hydrogen bonding and melting point. Furthermore, the structure of anhydrous sodium valproate was determined for the first time using 3DED. To address its sensitivity to both electrons and moisture, a glovebox was used to preserve the anhydrous structure during the cryo-sample preparation. Cryogenic cooling was then employed during data collection to reduce electron-beam damage.

Moreover, the structure of ZMQ-1, the first stable meso-microporous aluminosilicate zeolite, was uncovered using 3DED. To determine the position of the organic structure-directing agents (OSDAs), the low-dose cryo-cRED method was employed to stabilize OSDA molecules against electron-beam damage. Likewise, three isostructural aluminum(III)-monocarboxylates (CAU-71-X, where X = Ac, Prop, and TGA), were studied using low-dose cryo-cRED. This combined method stabilized the highly flexible ligands, enabling both structure determination and positional disorder refinement of the CAU-71 compounds.