Nanoscale imaging of living cells by liquid phase electron microscopy

Biocrystallization, the process by which cells form crystals, contributes to inflammatory and pathogenic diseases such as atherosclerosis, kidney stones, and gout, and underpins malaria pathogenesis, where frontline therapeutics directly inhibit crystal formation. Understanding how cells create crystals is therefore vital for developing strategies to inhibit their formation and prevent disease progression. However, these dynamic crystal nucleation and growth processes in living cells, taking place at the nanometer scale, cannot be captured with current high-resolution imaging methods that rely on freezing samples. Liquid-phase electron microscopy (LP-EM) is a recently established tool for studying reactions in hydrated conditions at nanometer resolution, and it has been applied to investigate dynamic processes in inorganic, organic, and biological systems. Yet, imaging biological processes at high resolution remains beyond reach, primarily due to the loss of enzymatic activity under repeated exposure to damaging radiation. LivEM will address this challenge by integrating multiple dose- and damage-reduction strategies to establish the first live-cell LP-EM workflow. The project will entail (1) the design of graphene-based enclosures that provide radical scavenging, (2) optimization of sparse imaging to minimize scanned pixels, and (3) use of flow and recovery strategies to allow radiolytic relaxation. Together, these measures will reduce the effective dose by ~150–200×, enabling real-time nanoscale visualization of crystal formation without exceeding enzymatic inactivation thresholds. Guanine crystallization in unicellular eukaryotes will serve as a controllable model system for developing and validating the methodology. By bridging the gap between static high-resolution imaging and dynamic biological function, LivEM will open new opportunities for understanding clinically relevant biocrystallization and inform the development of drug therapeutics.