Growth Kinetics of Single Polymer Particles in Solution via Active-Feedback 3D Tracking

Abstract

The ability to directly observe chemical reactions at the single-molecule and single-particle level has enabled the discovery of behaviors otherwise obscured by the ensemble averaging in bulk measurements. However powerful, a common restriction of these studies to date has been the absolute requirement to surface tether or otherwise immobilize the chemical reagent/reaction of interest. This constraint arose from a fundamental limitation of conventional microscopy techniques, which could not track molecules or particles rapidly diffusing in three dimensions, as occurs in solution. However, much chemistry occurs in the solution phase, leaving single-particle/-molecule analysis of this critical area of science beyond the scope of available technology. Here we report the first solution-phase studies and measurements of any chemical reaction at single-particle/-molecule level in freely diffusing solution. During chemical reaction, freely diffusing polymer particles (D ~ 10-12 m2/s) yielded single-particle 3D trajectories and real-time volumetric images that were analyzed to extract the growth rates of individual particles. These volumetric images show that the average growth rate is a poor representation of the true underlying variability in polymer-particle growth behavior. These data revealed statistically significant populations of faster- and slower-growing particles at different depths in the sample, showing emergent heterogeneity while particles are still in the solution phase. These results go against the prevailing premise that chemical processes freely diffusing in solution will exhibit uniform kinetics. These new understandings of mechanisms behind polymer growth variations bring about an exciting opportunity to control particle-size and plausibly molecular weight polydispersity by the rational design of conditions to dictate spatial growth gradients. We anticipate that these studies will launch a new field of solution-phase, nonensemble-averaged measurements of chemical reactions.

Combined online Bayesian and windowed estimation of background and signal localization facilitates active-feedback particle tracking in complex environments

“…12,13 Recently, 3D single-molecule active real-time tracking (3D-SMART) has been applied to track single fluorophore-labelled biomolecules (including DNA, proteins, and RNA) in glycerol/water solutions, the binding of fluorescent protein labelled lentiviral particles to live cells from the extracellular space, protein corona formation on nanoparticles, and the chemical kinetics of single polymer particles. [14][15][16][17][18] Collectively, single-particle active-feedback tracking techniques successfully follow single fluorophores in vitro but require multiply labeled bright slow-moving particles for tracking in live cells.…”