Revisiting Point FRAP to Quantitatively Characterize Anomalous Diffusion in Live Cells

Fluorescence recovery after photobleaching (FRAP) is widely used to interrogate diffusion and binding of proteins in live cells. Herein, we apply two-photon excited FRAP with a diffraction limited bleaching and observation volume to study anomalous diffusion of unconjugated green fluorescence protein (GFP) <i>in vitro</i> and in cells. Experiments performed on dilute solutions of GFP reveal that reversible fluorophore bleaching can be mistakenly interpreted as anomalous diffusion. We derive a reaction-diffusion FRAP model that includes reversible photobleaching, and demonstrate that it properly accounts for these photophysics. We then apply this model to investigate the diffusion of GFP in HeLa cells and polytene cells of <i>Drosophila</i> larval salivary glands. GFP exhibits anomalous diffusion in the cytoplasm of both cell types and in HeLa nuclei. Polytene nuclei contain optically resolvable chromosomes, permitting FRAP experiments that focus separately on chromosomal or interchrosomal regions. We find that GFP exhibits anomalous diffusion in chromosomal regions but diffuses normally in regions devoid of chromatin. This observation indicates that obstructed transport through chromatin and not crowding by macromolecules is a source of anomalous diffusion in polytene nuclei. This behavior is likely true in other cells, so it will be important to account for this type of transport physics and for reversible photobleaching to properly interpret future FRAP experiments on DNA-binding proteins

RNA Polymerase II Subunits Exhibit a Broad Distribution of Macromolecular Assembly States in the Interchromatin Space of Cell Nuclei

Nearly all cellular processes are enacted by multi-subunit protein complexes, yet the assembly mechanism of most complexes is not well understood. The anthropomorphism “protein recruitment” that is used to describe the concerted binding of proteins to accomplish a specific function conceals significant uncertainty about the underlying physical phenomena and chemical interactions governing the formation of macromolecular complexes. We address this deficiency by investigating the diffusion dynamics of two RNA polymerase II subunits, Rpb3 and Rpb9, in regions of live <i>Drosophila</i> cell nuclei that are devoid of chromatin binding sites. Using FRAP microscopy, we demonstrate that both unengaged subunits are incorporated into a broad distribution of complexes, with sizes ranging from free (unincorporated) proteins to those that have been predicted for fully assembled gene transcription units. In live cells, Rpb3 exhibits regions of stability at both size extremes connected by a continuous distribution of complexes. Corresponding measurements on cellular extracts reveal a distribution that retains peaks at the extremes but not in between, suggesting that partially assembled complexes are less stable. We propose that the broad distribution of macromolecular species allows for mechanistic flexibility in the assembly of transcription complexes

Power-Law Kinetics in the Photoluminescence of Dye-Sensitized Nanoparticle Films: Implications for Electron Injection and Charge Transport

Dye-sensitized solar cells have provided a model to inexpensively harness solar energy, but the underlying physics that limit their efficiency are still not well understood. We probe electron injection in sensitized nanocrystalline TiO₂ films using time-correlated single photon counting (TCSPC) to measure time-dependent chromophore photoluminescence quenching. The time-dependent emission exhibits kinetics that become faster and more dispersive with increasing ionic concentrations in both water and acetonitrile; we quantify these trends by fitting the data using several kinetic models. Even more notably, we show that the residual emission under conditions that favor efficient electron injection exhibits a power-law decay in time. We attribute this highly dispersive kinetic behavior to electron injection from the dye into localized acceptor states of the TiO₂ nanoparticle film, which exhibits a distribution of injection rate constants that depend on the energetic distribution of sub-band-gap trap states

Investigation of Factors That Affect Excited-State Lifetime Distribution of Dye-Sensitized Nanoparticle Films

We investigate the influence of potential determining ions and applied electric potentials on the excited-state lifetime distribution of sensitized TiO₂ nanoparticle films by using time-correlated single photon counting to measure the time-dependent photoluminescence decay. The data are consistent with quenching by excited-state electron injection into localized semiconductor acceptor states that are distributed in energy. We show that the characteristic lifetime and the amount of dispersion in the lifetime distribution exhibit a strong correlation that is the same for all of the chemical additives and for the applied bias conditions. The universal nature of this correlation under conditions that affect the distribution of available acceptor states differently may be due to the exponential form of the TiO₂ sub-bandgap density of states