Optical Monitoring of In Situ Iron Loading into Single, Native Ferritin Proteins.

Ferritin is a protein that stores and releases iron to prevent diseases associated with iron dysregulation in plants, animals, and bacteria. The conversion between iron-loaded holo-ferritin and empty apo-ferritin is an important process for iron regulation. To date, studies of ferritin have used either ensemble measurements to quantify the characteristics of a large number of proteins or single-molecule approaches to interrogate labeled or modified proteins. Here we demonstrate the first real-time study of the dynamics of iron ion loading and biomineralization within a single, unlabeled ferritin protein. Using optical nanotweezers, we trapped single apo- and holo-ferritins indefinitely, distinguished one from the other, and monitored their structural dynamics in real time. The study presented here deepens the understanding of the iron uptake mechanism of ferritin proteins, which may lead to new therapeutics for iron-related diseases.

Low-Power Sonication Can Alter Extracellular Vesicle Size and Properties.

Low-power sonication is widely used to disaggregate extracellular vesicles (EVs) after isolation, however, the effects of sonication on EV samples beyond dispersion are unclear. The present study analysed the characteristics of EVs collected from mesenchymal stem cells (MSCs) after sonication, using a combination of transmission electron microscopy, direct stochastic optical reconstruction microscopy, and flow cytometry techniques. Results showed that beyond the intended disaggregation effect, sonication using the lowest power setting available was enough to alter the size distribution, membrane integrity, and uptake of EVs in cultured cells. These results point to the need for a more systematic analysis of sonication procedures to improve reproducibility in EV-based cellular experiments.

Rapid and accurate analysis of stem cell-derived extracellular vesicles with super resolution microscopy and live imaging.

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20-30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication.