Single-molecule diffusivity quantification in egg extracts elucidates physicochemical properties of the cytoplasm.

Unlabelled: The living cell creates a unique internal molecular environment that is challenging to characterize. By combining single-molecule displacement/diffusivity mapping (SM M) with physiologically active extracts prepared from eggs, we sought to elucidate molecular properties of the cytoplasm. Quantification of the diffusion coefficients of 15 diverse proteins in extract showed that, compared to in water, negatively charged proteins diffused ∼50% slower, while diffusion of positively charged proteins was reduced by ∼80-90%.

Hypersensitivity of the vimentin cytoskeleton to net-charge states and Coulomb repulsion.

As with most intermediate filament systems, the hierarchical self-assembly of vimentin into nonpolar filaments requires no nucleators or energy input. Utilizing a set of live-cell, single-molecule, and super-resolution microscopy tools, here we show that in mammalian cells, the assembly and disassembly of the vimentin cytoskeleton is highly sensitive to the protein net charge state. Starting with the intriguing observation that the vimentin cytoskeleton fully disassembles under hypotonic stress yet reassembles within seconds upon osmotic pressure recovery, we pinpoint ionic strength as its underlying driving factor.

Single-Molecule Diffusivity Quantification Unveils Ubiquitous Net Charge-Driven Protein-Protein Interaction.

Recent microscopy and nuclear magnetic resonance (NMR) studies have noticed substantial suppression of intracellular diffusion for positively charged proteins, suggesting an overlooked role of electrostatic attraction in nonspecific protein interactions in a predominantly negatively charged intracellular environment. Utilizing single-molecule detection and statistics, here, we quantify in aqueous solutions how protein diffusion, in the limit of low diffuser concentration to avoid aggregate/coacervate formation, is modulated by differently charged interactor proteins over wide concentration ranges. We thus report substantially suppressed diffusion when oppositely charged interactors are added at parts per million levels, yet unvaried diffusivities when same-charge interactors are added beyond 1%.

A composite electrodynamic mechanism to reconcile spatiotemporally resolved exciton transport in quantum dot superlattices.

Quantum dot (QD) solids are promising optoelectronic materials; further advancing their device functionality requires understanding their energy transport mechanisms. The commonly invoked near-field Förster resonance energy transfer (FRET) theory often underestimates the exciton hopping rate in QD solids, yet no consensus exists on the underlying cause. In response, we use time-resolved ultrafast stimulated emission depletion (STED) microscopy, an ultrafast transformation of STED to spatiotemporally resolve exciton diffusion in tellurium-doped cadmium selenide-core/cadmium sulfide-shell QD superlattices.

Single-Molecule Displacement Mapping Indicates Unhindered Intracellular Diffusion of Small (≲1 kDa) Solutes.

While fundamentally important, the intracellular diffusion of small (≲1 kDa) solutes has been difficult to elucidate due to challenges in both labeling and measurement. Here we quantify and spatially map the translational diffusion patterns of small solutes in mammalian cells by integrating several recent advances. In particular, by executing tandem stroboscopic illumination pulses down to 400 μs separation, we extend single-molecule displacement/diffusivity mapping (SMdM), a super-resolution diffusion quantification tool, to small solutes with high diffusion coefficients of >300 μm/s.

Size-Dependent Suppression of Molecular Diffusivity in Expandable Hydrogels: A Single-Molecule Study.

By repurposing the recently popularized expansion microscopy to control the meshwork size of hydrogels, we examine the size-dependent suppression of molecular diffusivity in the resultant tuned hydrogel nanomatrices over a wide range of polymer fractions of ∼0.14-7 wt %. With our recently developed single-molecule displacement/diffusivity mapping (SMM) microscopy methods, we thus show that with a fixed meshwork size, larger molecules exhibit more impeded diffusion and that, for the same molecule, diffusion is progressively more suppressed as the meshwork size is reduced; this effect is more prominent for the larger molecules.

Single-molecule displacement mapping indicates unhindered intracellular diffusion of small (<~1 kDa) solutes.

While fundamentally important, the intracellular diffusion of small (<~1 kDa) solutes has been difficult to elucidate due to challenges in both labeling and measurement. Here we quantify and spatially map the translational diffusion patterns of small solutes in mammalian cells by integrating several recent advances. In particular, by executing tandem stroboscopic illumination pulses down to 400-μs separation, we extend single-molecule displacement/diffusivity mapping (SM M), a super-resolution diffusion quantification tool, to small solutes with high diffusion coefficients of >300 μm /s.

Fluorogenic Dimers as Bright Switchable Probes for Enhanced Super-Resolution Imaging of Cell Membranes.

Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualizing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes, the single-molecule localization is hampered by the fast lateral diffusion of membrane probes.

Displacement Statistics of Unhindered Single Molecules Show no Enhanced Diffusion in Enzymatic Reactions.

Recent studies have sparked debate over whether catalytic reactions enhance the diffusion coefficients of enzymes. Through high statistics of the transient (600 μs) displacements of unhindered single molecules freely diffusing in common buffers, we here quantify for four enzymes under catalytic turnovers. We thus formulate how ∼ ±1% precisions may be achieved for , and show no changes in diffusivity for catalase, urease, aldolase, and alkaline phosphatase under the application of wide concentration ranges of substrates.

Systematic Analysis of Fatty Acids in Human Cells with a Multiplexed Isobaric Tag (TMT)-Based Method.

Fatty acids (FAs) are essential components in cells and are involved in many cellular activities. Abnormal FA metabolism has been reported to be related to human diseases such as cancer and cardiovascular diseases. Identification and quantification of FAs provide insights into their functions in biological systems, but it is very challenging to analyze them due to their structures and properties.