Emergence and Rearrangement of Dynamic Supramolecular Aggregates Visualized by Interferometric Scattering Microscopy

Abstract

Studying dynamic self-assembling systems in their native environment is essential for understanding the mechanisms of self-assembly and thereby exerting full control over these processes. Traditional ensemble-based analysis methods often struggle to reveal critical features of the self-assembly that occur at the single particle level. Here, we describe a label-free single-particle assay to visualize real-time self-assembly in aqueous solutions by interferometric scattering microscopy. We demonstrate how the assay can be applied to biphasic reactions yielding micellar or vesicular aggregates, detecting the onset of aggregate formation, quantifying the kinetics at the single particle level, and distinguishing sigmoidal and exponential growth of aggregate populations. Furthermore, we can follow the evolution in aggregate size in real time, visualizing the nucleation stages of the self-assembly processes and record phenomena such as incorporation of oily components into the micelle or vesicle lumen.

Harnessing autocatalytic reactions in polymerization and depolymerization

“…[63] Biphasic thiol-X reactions either on the microscope coverslip or under traditional batch conditions were observed in situ by this label-free microscopic technique, which relies on the induced scattering properties changing as the result of the non-specific binding of aggregates to the glass surface. iSCAT observations were corroborated with measurements acquired from fluorescence spectrometry [72] as well as ultra-performance liquid chromatography (UPLC). [74] More importantly, iSCAT exhibited decisive advantages (due to its high sensitivity) over dynamic light scattering (DLS) for allowing verification of the nucleation stage as the reaction proceeded.…”

“…Recently, an alternative optical microscopic technique-interferometric scattering microscopy (iSCAT)was extensively reported by Fletcher and collaborators for studying biphasic physical autocatalysis. [63,72] iSCAT allows direct visualization of nano-sized micelles as well as vesicles. As compared to the thiol in the first reported autocatalytic thiol-ene reaction [73] (Fig.…”

Coupled Metabolic Cycles Allow Out‐of‐Equilibrium Autopoietic Vesicle Replication

“…In order to study the kinetics of the formation of the aggregates by iSCAT, we have taken aliquots throughout the reaction, and counted the number of particles binding to the glass in each aliquot. [27] All experiments showed a sigmoidal profile, with a clear lag period when no particles were detected, followed by growth in the population of aggregates (Figure 2 c, top). These results are in good agreement with those obtained by following the concentration of 7 by UPLC (Figure 3 c, bottom), suggesting that the aggregates form as soon as detectable amount of surfactant is present.…”

“…Such distribution is characteristic of vesicles, as compared to nearly symmetric distributions with much lower contrast we have previously observed for micelles (Supporting Information Table S5.1). [27] Significant differences in the peak, median and mean of the distribution suggest that the aggregate population is polydisperse, and there is substantial variation in the size of the particles that are present in solution. Our observation of a highly polydisperse mixture was confirmed by DLS measurements, showing a significantly broadened size distribution.…”

Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution

“…2) to room temperature and thaw the proteins in hand (keep them on ice afterward). NOTE: iSCAT shows the signal of some detergents and small molecules that resemble protein signals 45 . DTT is one of those small molecules, and that is why it is not used for this experiment.…”