Size determination of lipid nanoparticles on a single-particle level using laser scanning microscopy

Lipid nanoparticles (LNPs) are becoming increasingly important for the delivery of nucleic acids, such as mRNA or siRNA, into cells. Different ensemble-based techniques already exist, but single-particle level characterization is crucial to improve our understanding of composition and loading efficiency of often heterogeneous LNP samples. In order to perform single-particle size characterization a laser-scanning microscopy setup is realized with a custom-built piezo stage. To determine the position of individual, freely moving LNPs, a focused laser beam is scanned across a regular grid, and the fluorescence at each grid location is analyzed. Using a feedback algorithm, the piezo stage tracks individual particles for several seconds. In post-processing particle trajectories are reconstructed, from which diffusion coefficients and sizes are estimated. We applied the method to DiOlabeled LNPs, as well as quantum dots as small as 15 nm, indicating that we are able to analyze small particles through fast piezo tracking.