TEM of Biologically Induced Reactions: Kidney Stone Mineralization and E. coli-Fiber Interactions

The recent development of liquid electron microscopy (EM) has enabled real-time studies of biological and biologically relevant species at the nanoscale. Here, liquid transmission EM (TEM) provides fundamental, mechanistic insight into: 1) the nanoscale interaction of bacteria with antibacterial surface topologies, and 2) into the nanoscale nucleation and dissolution of calcium oxalate (CaOx), which is the major phase of approximately 80% of kidney stones. In contrast to conventional antibiotics or chemical treatments, nanoscale surface roughness can kill bacteria or prevent bacterial binding without the presence of toxic compounds. Rough surface topologies were first etched on biocompatible poly (lactic-glycolic acid) (PLGA) microparticles (MPs). In contrast to previous mathematical models, liquid TEM of E. coli and PLGA MPs revealed that protrusions on the surface of the MPs directly penetrate the bacteria to produce cell lysis. Additionally, the nucleation and formation of CaOx was observed in liquid TEM. CaOx crystallizes via the classical nucleation pathway or via a non-classical multiphase nucleation pathway. The classical nucleation pathway forms rhombohedral CaOx monohydrate (COM), while the non-classical multiphase pathway forms square CaOx (COM). The presence of citrate, a kidney stone inhibitor, alters CaOx nucleation in two ways: first, at low concentrations of CaOx, calcium and citrate form soluble interactions which prevent the nucleation of CaOx. At higher CaOx concentrations, CaOx overcomes the effect of citrate. Citrate instead draws more water molecules into the formed CaOx particle. This increases the hydration state to form dihydrate (COD). Previous studies indicated that COD does not bind to cell walls as well as COM binds to cell walls. Thus, citrate prevents kidney stones by preventing the nucleation of CaOx and by promoting the excretion of COD. Liquid TEM was supported by extensive molecular dynamics (MD) simulations and ex situ benchtop experiments.