Multipair Förster Resonance Energy Transfer via Spectrally Resolved Single-Molecule Detection.

Förster resonance energy transfer (FRET) is a powerful tool for studying molecular interactions. Its use for studying interactions involving more than two molecules, however, has been limited by spectral crosstalk among the fluorophores. Here, we report multispectral FRET (msFRET) for imaging multiple pairs of interactions in parallel by spectrally resolving single fluorescent molecules. By using a dual (positional and spectral) channel and wide-field imaging configuration, fluorophores with emission maxima as close as 6-10 nm could be reliably distinguished. We demonstrate msFRET by continuously monitoring the hybridization dynamics among 2 × 2 pairs of DNA oligos in parallel using Cy3 and Cy3.5 as donors and Cy5 and Cy5.5 as acceptors. Aside from studying molecular interactions, msFRET may also find applications in probing fluorophore photophysics during FRET and in multiplexed superresolution imaging.

The design and evaluation of a novel algorithm for automated preference card optimization.

BACKGROUND: Inaccurate surgical preference cards (supply lists) are associated with higher direct costs, waste, and delays. Numerous preference card improvement projects have relied on institution-specific, manual approaches of limited reproducibility. We developed and tested an algorithm to facilitate the first automated, informatics-based, fully reproducible approach. METHODS: The algorithm cross-references the supplies used in each procedure and listed on each preference card and uses a time-series regression to estimate the likelihood that each quantity listed on the preference card is inaccurate. Algorithm performance was evaluated by measuring changes in direct costs between preference cards revised with the algorithm and preference cards that were not revised or revised without use of the algorithm. Results were evaluated with a difference-in-differences (DID) multivariate fixed-effects model of costs during an 8-month pre-intervention and a 15-month post-intervention period. RESULTS: The accuracies of the quantities of 469 155 surgeon-procedure-specific items were estimated. Nurses used these estimates to revise 309 preference cards across eight surgical services corresponding to, respectively, 1777 and 3106 procedures in the pre- and post-intervention periods. The average direct cost of supplies per case decreased by 8.38% ($352, SD $6622) for the intervention group and increased by 13.21% ($405, SD $14 706) for the control group (P < .001). The DID analysis showed significant cost reductions only in the intervention group during the intervention period (P < .001). CONCLUSION: The optimization of preference cards with a variety of institution-specific, manually intensive approaches has led to cost savings. The automated algorithm presented here produced similar results that may be more readily reproducible.

Author Correction: Fast and multiplexed superresolution imaging with DNA-PAINT-ERS.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Fast and multiplexed superresolution imaging with DNA-PAINT-ERS.

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) facilitates multiplexing in superresolution microscopy but is practically limited by slow imaging speed. To address this issue, we propose the additions of ethylene carbonate (EC) to the imaging buffer, sequence repeats to the docking strand, and a spacer between the docking strand and the affinity agent. Collectively termed DNA-PAINT-ERS (E = EC, R = Repeating sequence, and S = Spacer), these strategies can be easily integrated into current DNA-PAINT workflows for both accelerated imaging speed and improved image quality through optimized DNA hybridization kinetics and efficiency. We demonstrate the general applicability of DNA-PAINT-ERS for fast, multiplexed superresolution imaging using previously validated oligonucleotide constructs with slight modifications.

High-throughput, single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking.

Membrane nanodomains have been implicated in Ras signaling, but what these domains are and how they interact with Ras remain obscure. Here, using single particle tracking with photoactivated localization microscopy (spt-PALM) and detailed trajectory analysis, we show that distinct membrane domains dictate KRasG12D (an active KRas mutant) diffusion and trafficking in U2OS cells. KRasG12D exhibits an immobile state in ~70 nm domains, each embedded in a larger domain (~200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. Moreover, KRasG12D is continuously removed from the membrane via the immobile state and replenished to the fast state, reminiscent of Ras internalization and recycling. Importantly, both the diffusion and trafficking properties of KRasG12D remain invariant over a broad range of protein expression levels. Our results reveal how membrane organization dictates membrane diffusion and trafficking of Ras and offer new insight into the spatial regulation of Ras signaling.

The Ras family of proteins play an important role in relaying signals from the outside to the inside of the cell. Ras proteins are attached by a fatty tail to the inner surface of the cell membrane. When activated they transmit a burst of signal that controls critical behaviors like growth, survival and movement. It has been suggested that to prevent these signals from being accidently activated, Ras molecules must group together at specialized sites within the membrane before passing on their message. However, visualizing how Ras molecules cluster together at these domains has thus far been challenging. As a result, little is known about where these sites are located and how Ras molecules come to a stop at these domains. Now, Lee et al. have combined two microscopy techniques called 'single-particle tracking' and 'photoactivated localization microscopy' to track how individual molecules of activated Ras move in human cells grown in the lab. This revealed that Ras molecules quickly diffuse along the inside of the membrane until they arrive at certain locations that cause them to halt. However, computer models consisting of just the 'fast' and 'immobile' state could not correctly re-capture the way Ras molecules moved along the membrane. Lee et al. found that for these models to mimic the movement of Ras, a third 'intermediate' state of Ras mobility needed to be included. To investigate this further, Lee et al. created a fluorescent map that overlaid all the individual paths taken by each Ras molecule. The map showed regions in the membrane where the Ras molecules had stopped and possibly clustered together. Each of these 'immobilization domains' were then surrounded by an 'intermediate domain' where Ras molecules had begun to slow down their movement. Although the intermediate domains did not last long, they seemed to guide Ras molecules into the immobilization domains where they could cluster together with other molecules. From there, the cell constantly removed Ras molecules from these membrane domains and returned them back to their 'fast' diffusing state. Mutations in Ras proteins occur in around a third of all cancers, so a better understanding of their dynamics could help with future drug discovery. The methods used here could also be used to investigate the movement of other signaling molecules.

Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging.

Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels. Images of the same molecule in the two channels are then combined to determine both the location and the identity of the molecule. The single-objective configuration of our approach allows for flexible sample geometry and the use of a live-cell incubation chamber required for live-cell SMT. Despite a lower photon yield, we achieve excellent spatial (20-40 nm) and spectral (10-15 nm) resolutions comparable to those obtained with dual-objective, spectrally resolved Stochastic Optical Reconstruction Microscopy. Furthermore, motions of the fluorescent molecules did not cause loss of spectral resolution owing to the dual-channel spectral calibration. We demonstrate SMT in three (and potentially more) colors using spectrally proximal fluorophores and single-laser excitation, and show that trajectories of each species can be reliably extracted with minimal cross talk.

Using microsecond single-molecule FRET to determine the assembly pathways of T4 ssDNA binding protein onto model DNA replication forks.

DNA replication is a core biological process that occurs in prokaryotic cells at high speeds (∼1 nucleotide residue added per millisecond) and with high fidelity (fewer than one misincorporation event per 107 nucleotide additions). The ssDNA binding protein [gene product 32 (gp32)] of the T4 bacteriophage is a central integrating component of the replication complex that must continuously bind to and unbind from transiently exposed template strands during DNA synthesis. We here report microsecond single-molecule FRET (smFRET) measurements on Cy3/Cy5-labeled primer-template (p/t) DNA constructs in the presence of gp32. These measurements probe the distance between Cy3/Cy5 fluorophores that label the ends of a short (15-nt) segment of ssDNA attached to a model p/t DNA construct and permit us to track the stochastic interconversion between various protein bound and unbound states. The length of the 15-nt ssDNA lattice is sufficient to accommodate up to two cooperatively bound gp32 proteins in either of two positions. We apply a unique multipoint time correlation function analysis to the microsecond-resolved smFRET data obtained to determine and compare the kinetics of various possible reaction pathways for the assembly of cooperatively bound gp32 protein onto ssDNA sequences located at the replication fork. The results of our analysis reveal the presence and translocation mechanisms of short-lived intermediate bound states that are likely to play a critical role in the assembly mechanisms of ssDNA binding proteins at replication forks and other ss duplex junctions.

Using Multiorder Time-Correlation Functions (TCFs) To Elucidate Biomolecular Reaction Pathways from Microsecond Single-Molecule Fluorescence Experiments.

Recent advances in single-molecule fluorescence imaging have made it possible to perform measurements on microsecond time scales. Such experiments have the potential to reveal detailed information about the conformational changes in biological macromolecules, including the reaction pathways and dynamics of the rearrangements involved in processes, such as sequence-specific DNA "breathing" and the assembly of protein-nucleic acid complexes. Because microsecond-resolved single-molecule trajectories often involve "sparse" data, that is, they contain relatively few data points per unit time, they cannot be easily analyzed using the standard protocols that were developed for single-molecule experiments carried out with tens-of-millisecond time resolution and high "data density." Here, we describe a generalized approach, based on time-correlation functions, to obtain kinetic information from microsecond-resolved single-molecule fluorescence measurements. This approach can be used to identify short-lived intermediates that lie on reaction pathways connecting relatively long-lived reactant and product states. As a concrete illustration of the potential of this methodology for analyzing specific macromolecular systems, we accompany the theoretical presentation with the description of a specific biologically relevant example drawn from studies of reaction mechanisms of the assembly of the single-stranded DNA binding protein of the T4 bacteriophage replication complex onto a model DNA replication fork.

Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions.

DNA "breathing" is a thermally driven process in which base-paired DNA sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded DNA coding templates located in the double-stranded DNA "interior," and it is likely that fluctuations of the sugar-phosphate backbones of dsDNA that result in mechanistically useful local base pair opening reactions can be exploited by such DNA regulatory proteins. Such motions are difficult to observe in bulk measurements, both because they are infrequent and because they often occur on microsecond time scales that are not easy to access experimentally. We report single-molecule fluorescence experiments with polarized light, in which tens-of-microseconds rotational motions of internally labeled iCy3/iCy5 donor-acceptor Förster resonance energy transfer fluorophore pairs that have been rigidly inserted into the backbones of replication fork constructs are simultaneously detected using single-molecule Förster resonance energy transfer and single-molecule fluorescence-detected linear dichroism signals. Our results reveal significant local motions in the ∼100-µs range, a reasonable time scale for DNA breathing fluctuations of potential relevance for DNA-protein interactions. Moreover, we show that both the magnitudes and the relaxation times of these backbone breathing fluctuations are significantly perturbed by interactions of the fork construct with a nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting that these motions may play a fundamental role in the initial binding, assembly, and function of the processive helicase-primase (primosome) component of the bacteriophage T4-coded DNA replication complex.

A single-molecule view of the assembly pathway, subunit stoichiometry, and unwinding activity of the bacteriophage T4 primosome (helicase-primase) complex.

Single-molecule fluorescence resonance energy transfer (smFRET) methods were used to study the assembly pathway and DNA unwinding activity of the bacteriophage T4 helicase-primase (primosome) complex. The helicase substrates used were surface-immobilized model DNA replication forks "internally" labeled in the duplex region with opposed donor/acceptor (iCy3/iCy5) chromophore pairs in the lagging and leading strands. The time dependence of the smFRET signals was monitored during the unwinding process, and helicase rates and processivities were measured as a function of GTP concentration. This smFRET approach was also used to investigate the subunit stoichiometry of the primosome and the assembly pathway required to form functional and fully active primosome-DNA complexes. We confirmed that gp41 helicase monomer subunits form stable hexameric helicases in the presence of GTP and that the resulting (gp41)(6) complexes bind only weakly at DNA fork junctions. The addition of a single subunit of gp61 primase stabilized the resulting primosome complex at the fork and resulted in fully active and processive primosome helicases with gp41:gp61 subunit ratios of 6:1, while higher and lower subunit ratios substantially reduced the primosome unwinding activity. The use of alternative assembly pathways resulted in a loss of helicase activity and the formation of metastable DNA-protein aggregates, which were easily detected in our smFRET experiments as intense light-scattering foci. These single-molecule experiments provide a detailed real-time visualization of the assembly pathway and duplex DNA unwinding activity of the T4 primosome and are consistent with more indirect equilibrium and steady state results obtained in bulk solution studies.

Ultrafast coherent electron spin flip in a modulation-doped CdTe quantum well.

We report the experimental realization of coherent electron spin flip in a modulation-doped CdTe quantum well. Coherent spin rotation is realized with an off-resonant laser pulse, which induces a polarization-dependent optical Stark shift in the trion resonance. Complete electron spin flip is made possible by a laser pulse designed to avoid excessive excitations of nearby exciton resonances and minimizes detrimental many-body effects. These results demonstrate an effective approach for ultrafast optical spin control in a complex spin system.