Artifact-free high-density localization microscopy analysis.

High-density analysis methods for localization microscopy increase acquisition speed but produce artifacts. We demonstrate that these artifacts can be eliminated by the combination of Haar wavelet kernel (HAWK) analysis with standard single-frame fitting. We tested the performance of this method on synthetic, fixed-cell, and live-cell data, and found that HAWK preprocessing yielded reconstructions that reflected the structure of the sample, thus enabling high-speed, artifact-free super-resolution imaging of live cells.

Low power super resolution fluorescence microscopy by lifetime modification and image reconstruction.

We demonstrate a new method for obtaining sub-diffraction resolution in fluorescence microscopy. The technique involves the analysis of the time evolution of fluorescence images in the presence of weak and unstructured (fundamental Gaussian) continuous wave stimulated emission depletion. A reduced point spread functions (PSF) is obtained by the recombination of time segments of the evolving image. A significant reduction in the PSF for 20 nm fluorescent beads (ca. 240 nm to 125 nm) is obtained with an on-sample power of 7.5 mW (17 MW/cm2) - substantially lower than that required for spatially structured stimulated emission depletion microscopy.

Restricted state selection in fluorescent protein Förster resonance energy transfer.

The measurement of donor lifetime modification by Förster resonance energy transfer (FRET) is a widely used tool for detecting protein-protein interactions and protein conformation change. Such measurements can be compromised by the presence of a significant noninteracting fraction of molecules. Combining time-resolved intensity and anisotropy measurements gives access to both molecular distance and orientation. Fluorescent proteins frequently used to detect energy transfer in biological systems often exhibit decay characteristics indicative of more than one excited state. However, little attention has thus far been given to the specific modes of energy transfer, in particular, which states are predominantly coupled. Here, we use a previously characterized dimerization system to study energy transfer between EGFP and mCherry. Optically excited EGFP and mCherry both exhibit biexponential decays, and FRET should therefore involve dipole-dipole transfer between these four states. Analysis of the sensitized fluorescence anisotropy and intensity decays indicates that FRET transfer is predominantly from the shorter lived EGFP emitting state (2.43 ns) to the longer lived (ca. 2.77 ns) minority component (ca. 16%) of the optically excited mCherry emission. This high degree of state selection between these two widely used FRET pairs highlights the fundamental differences that can arise between direct optical excitation of an isotropic molecular population and dipole-dipole coupling in a far from isotropic interaction geometry and has consequences regarding the accurate interpretation of fluorescent protein FRET data.

State-to-state, multi-collision, energy transfer in H-H2 gas ensembles.

We use our recently developed computational model of energy flow in gas ensembles to study translation-to-internal energy conversion in an ensemble consisting of H2(0; 0) in a bath of H atoms. This mixture is found in plasmas of industrial importance and also in interstellar clouds. The storage of energy of relative motion as rovibrational energy of H2 represents a potential mechanism for cooling translation. This may have relevance in astrophysical contexts such as the post-recombination epoch of the early universe when hydrogenic species dominated and cooling was a precondition for the formation of structured objects. We find that conversion of translational motion to H2 vibration and rotation is fast and, in our closed system, is complete within around 100 cycles of ensemble collisions. Large amounts of energy become stored as H2 vibration and a tentative mechanism for this unequal energy distribution is suggested. The "structured dis-equilibrium" we observe is found to persist through many collision cycles. In contrast to the rapidity of excitation, the relaxation of H2(6; 10) in H is very slow and not complete after 10(5) collision cycles. The quasi-equilibrium modal temperatures of translation, rotation, and vibration are found to scale linearly with collision energy but at different rates. This may be useful in estimating the partitioning of energy within a given H + H2 ensemble.

Competitive partitioning of rotational energy in gas ensemble equilibration.

A wide-ranging computational study of equilibration in binary mixtures of diatomic gases reveals the existence of competition between the constituent species for the orbital angular momentum and energy available on collision with the bath gas. The ensembles consist of a bath gas AB(v;j), and a highly excited minor component CD(v';j'), present in the ratio AB:CD = 10:1. Each ensemble contains 8000 molecules. Rotational temperatures (T(r)) are found to differ widely at equilibration with T(r)(AB)/T(r)(CD) varying from 2.74 to 0.92, indicating unequal partitioning of rotational energy and angular momentum between the two species. Unusually, low values of T(r) are found generally to be associated with diatomics of low reduced mass. To test effects of the equi-partition theorem on low T(r) we undertook calculations on HF(6;4) in N(2)(0;10) over the range 100-2000 K. No significant change in T(r)(N2)/T(r)(HF) was found. Two potential sources of rotational inequality are examined in detail. The first is possible asymmetry of -Δj and +Δj probabilities for molecules in mid- to high j states resulting from the quadratic dependence of rotational energy on j. The second is the efficiency of conversion of orbital angular momentum, generated on collision with bath gas molecules, into molecular rotation. Comparison of these two possible effects with computed T(r)(AB)/T(r)(CD) shows the efficiency factor to be an excellent predictor of partitioning between the two species. Our finding that T(r) values for molecules such as HF and OH are considerably lower than other modal temperatures suggests that the determination of gas ensemble temperatures from Boltzmann fits to rotational distributions of diatomics of low reduced mass may require a degree of caution.

Multiple conformations of full-length p53 detected with single-molecule fluorescence resonance energy transfer.

The tumor suppressor p53 is a member of the emerging class of proteins that have both folded and intrinsically disordered domains, which are a challenge to structural biology. Its N-terminal domain (NTD) is linked to a folded core domain, which has a disordered link to the folded tetramerization domain, which is followed by a disordered C-terminal domain. The quaternary structure of human p53 has been solved by a combination of NMR spectroscopy, electron microscopy, and small-angle X-ray scattering (SAXS), and the NTD ensemble structure has been solved by NMR and SAXS. The murine p53 is reported to have a different quaternary structure, with the N and C termini interacting. Here, we used single-molecule FRET (SM-FRET) and ensemble FRET to investigate the conformational dynamics of the NTD of p53 in isolation and in the context of tetrameric full-length p53 (flp53). Our results showed that the isolated NTD was extended in solution with a strong preference for residues 66-86 forming a polyproline II conformation. The NTD associated weakly with the DNA binding domain of p53, but not the C termini. We detected multiple conformations in flp53 that were likely to result from the interactions of NTD with the DNA binding domain of each monomeric p53. Overall, the SM-FRET results, in addition to corroborating the previous ensemble findings, enabled the identification of the existence of multiple conformations of p53, which are often averaged and neglected in conventional ensemble techniques. Our study exemplifies the usefulness of SM-FRET in exploring the dynamic landscape of multimeric proteins that contain regions of unstructured domains.

Nuclear fascin regulates cancer cell survival.

Fascin is an important regulator of F-actin bundling leading to enhanced filopodia assembly. Fascin is also overexpressed in most solid tumours where it supports invasion through control of F-actin structures at the periphery and nuclear envelope. Recently, fascin has been identified in the nucleus of a broad range of cell types but the contributions of nuclear fascin to cancer cell behaviour remain unknown. Here, we demonstrate that fascin bundles F-actin within the nucleus to support chromatin organisation and efficient DDR. Fascin associates directly with phosphorylated Histone H3 leading to regulated levels of nuclear fascin to support these phenotypes. Forcing nuclear fascin accumulation through the expression of nuclear-targeted fascin-specific nanobodies or inhibition of Histone H3 kinases results in enhanced and sustained nuclear F-actin bundling leading to reduced invasion, viability, and nuclear fascin-specific/driven apoptosis. These findings represent an additional important route through which fascin can support tumourigenesis and provide insight into potential pathways for targeted fascin-dependent cancer cell killing.

Equilibration of vibrationally excited OH in atomic and diatomic bath gases.

In this work, a computational model of state-to-state energy flow in gas ensembles is used to investigate collisional relaxation of excited OH, present as a minor species in various bath gases. Rovibrational quantum state populations are computed for each component species in ensembles consisting of 8000 molecules undergoing cycles of binary collisions. Results are presented as quantum state populations and as (approximate) modal temperatures for each species after each collision cycle. Equilibration of OH is slow with Ar as the partner but much faster when N(2) and/or O(2) forms the bath gas. This accelerated thermalization is shown to be the result of near-resonant vibration-vibration transfer, with vibrational de-excitation in OH matched in energy by excitation in bath molecules. Successive near-resonant events result in an energy cascade. Such processes are highly dependent on molecule pair and on initial OH vibrational state. OH rotational temperatures initially increase, but at equilibration, they are lower than those of other modes. Possible reasons for this observation in molecules such as OH are suggested. There are indications of an order of precedent in the equilibration process, with vibrations taking priority over rotations, and potential explanations for this phenomenon are discussed.

Quantum state-resolved energy redistribution in gas ensembles containing highly excited N2.

A computational model is used to quantify the evolution of quantum state populations as highly vibrationally excited (14)N(2) ((14)N(2)∗) equilibrates in various bath gases. Multicollision energy disposal follows general principles established in related single collision processes. Thus when state-to-state routes permit, maximum amounts of energy are deposited into partner species by direct vibration-to-vibration (V-V) exchange. When these pathways are absent, e.g., when Ar is the bath species, relaxation is very slow and multistaged. Conversely, in a bath of v = 0 (14)N(2) molecules, 16 vibrational quanta (Δv = ± 8) are resonantly exchanged from (v;j) = (8;10) with vibrational equilibration so rapid that rotation and translation still lag far behind after 1000 collisions. Near-resonant V-V exchange dominates the initial phase when (15)N(2) forms the bath gas and although some rotational warming occurs, vibrational modes remain decoupled from, and significantly hotter than, the low heat capacity modes. These forms of behavior seem likely to characterize excited and bath species that have closely similar vibration and rotation constants. More generic in nature is (14)N(2) in O(2) or in a mixture that closely resembles air. Here, asymmetric V-V exchange is a dominant early feature in ensemble evolution but energy differences in the key vibration and rotation quanta lead to V-V energy defects that are compensated for by the low energy modes. This results in much more rapid ensemble equilibration, generally within 400-500 collisions, when O(2) is present even as a minor constituent. Our results are in good general agreement with those obtained from experimental studies of N(2) plasmas both in terms of modal temperatures and initial (first collision cycle) cross-sections.

Sub-diffraction error mapping for localisation microscopy images.

Assessing the quality of localisation microscopy images is highly challenging due to the difficulty in reliably detecting errors in experimental data. The most common failure modes are the biases and errors produced by the localisation algorithm when there is emitter overlap. Also known as the high density or crowded field condition, significant emitter overlap is normally unavoidable in live cell imaging. Here we use Haar wavelet kernel analysis (HAWK), a localisation microscopy data analysis method which is known to produce results without bias, to generate a reference image. This enables mapping and quantification of reconstruction bias and artefacts common in all but low emitter density data. By avoiding comparisons involving intensity information, we can map structural artefacts in a way that is not adversely influenced by nonlinearity in the localisation algorithm. The HAWK Method for the Assessment of Nanoscopy (HAWKMAN) is a general approach which allows for the reliability of localisation information to be assessed.

Modeling disequilibrium in gas ensembles: how quantum state populations evolve under multicollision conditions; CO*+Ar, CO, O2, and N2.

The method of Marsh and McCaffery [J. Chem. Phys. 117, 503 (2002)] is used to quantify how rovibrational populations and mode temperatures change as an ensemble of CO molecules, initially excited to (v;j)=(8;12), evolves to thermal equilibrium in a bath gas. The bath gases considered are Ar, N(2), O(2), and CO all at 300 K with the diatomics in their (0;8) rovibrational states. Ensembles generally contain 1000 molecules, 10% of which are excited CO (CO( *)) molecules. State (v;j) populations and mode temperatures of CO* and bath molecules are calculated for successive collisions to 1000 or more. We find that relaxation to local thermodynamic equilibrium occurs in distinct phases that vary widely in rate of cooling. There is especially fast vibration-vibration (VV) exchange in CO*-CO mixtures that is largely decoupled from rotation and translation. Several aspects of ensemble behavior may be rationalized using concepts established in quantum state resolved single collision studies. We demonstrate the existence of a simultaneous energy quasiresonant, angular momentum conserving, low Deltaj VV process that can cause either ultrafast relaxation or up pumping of the kind seen in a number of experiments.

Combined AFM and super-resolution localisation microscopy: Investigating the structure and dynamics of podosomes.

Podosomes are mechanosensitive attachment/invasion structures that form on the matrix-adhesion interface of cells and protrude into the extracellular matrix to probe and remodel. Despite their central role in many cellular processes, their exact molecular structure and function remain only partially understood. We review recent progress in molecular scale imaging of podosome architecture, including our newly developed localisation microscopy technique termed HAWK which enables artefact-free live-cell super-resolution microscopy of podosome ring proteins, and report new results on combining fluorescence localisation microscopy (STORM/PALM) and atomic force microscopy (AFM) on one setup, where localisation microscopy provides the location and dynamics of fluorescently labelled podosome components, while the spatial variation of stiffness is mapped with AFM. For two-colour localisation microscopy we combine iFluor-647, which has previously been shown to eliminate the need to change buffer between imaging modes, with the photoswitchable protein mEOS3.2, which also enables live cell imaging.

FMNL2 regulates dynamics of fascin in filopodia.

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.

TNFR1 membrane reorganization promotes distinct modes of TNFα signaling.

Signaling by the ubiquitously expressed tumor necrosis factor receptor 1 (TNFR1) after ligand binding plays an essential role in determining whether cells exhibit survival or death. TNFR1 forms distinct signaling complexes that initiate gene expression programs downstream of the transcriptional regulators NFκB and AP-1 and promote different functional outcomes, such as inflammation, apoptosis, and necroptosis. Here, we investigated the ways in which TNFR1 was organized at the plasma membrane at the nanoscale level to elicit different signaling outcomes. We confirmed that TNFR1 forms preassembled clusters at the plasma membrane of adherent cells in the absence of ligand. After trimeric TNFα binding, TNFR1 clusters underwent a conformational change, which promoted lateral mobility, their association with the kinase MEKK1, and activation of the JNK/p38/NFκB pathway. These phenotypes required a minimum of two TNFR1-TNFα contact sites; fewer binding sites resulted in activation of NFκB but not JNK and p38. These data suggest that distinct modes of TNFR1 signaling depend on nanoscale changes in receptor organization.

Two-photon transitions in quadrupolar and branched chromophores: experiment and theory.

A combined experimental and theoretical study is conducted on a series of model compounds in order to assess the combined role of branching and charge symmetry on absorption, photoluminescence, and two-photon absorption (TPA) properties. The main issue of this study is to examine how branching of quadrupolar chomophores can lead to different consequences as compared to branching of dipolar chromophores. Hence, three structurally related pi-conjugated quadrupolar chromophores symmetrically substituted with donor end groups and one branched structure built from the assembly of three quadrupolar branches via a common donor moiety are used as model compounds. Their photophysical properties are studied using UV-vis spectroscopy, and the TPA spectra are determined through two-photon excited fluorescence experiments using femtosecond pulses in the 500-1000 nm range. Experimental studies are complemented by theoretical calculations. The applied theoretical methodology is based on time-dependent density functional theory, the Frenkel exciton model, and analysis in terms of the natural transition orbitals of relevant electronic states. Theory reveals that a symmetrical intramolecular charge transfer from the terminal donating groups to the middle of the molecule takes place in all quadrupolar chromophores upon photoexcitation. In contrast, branching via a central electron-donating triphenylamine moiety breaks the quadrupolar symmetry of the branches. Consequently, all Frank-Condon excited states have significant asymmetric multidimensional charge-transfer character upon excitation. Subsequent vibrational relaxation of the branched chromophore in the excited state leads to a localization of the excitation and fluorescence stemming from a single branch. As opposed to what was earlier observed when dipolar chromophores are branched via the same common electron-donating moiety, we find only a slight enhancement of the maximum TPA response of the branched compound with respect to an additive contribution of its quadrupolar branches. In contrast, substantial modifications of the spectral shape are observed. This is attributed to the subtle interplay of interbranch electronic coupling and asymmetry caused by branching.

Investigating State Restriction in Fluorescent Protein FRET Using Time-Resolved Fluorescence and Anisotropy.

Most fluorescent proteins exhibit multiexponential fluorescence decays, indicating a heterogeneous excited state population. FRET between fluorescent proteins should therefore involve multiple energy transfer pathways. We recently demonstrated the FRET pathways between EGFP and mCherry (mC), upon the dimerization of 3-phosphoinositide dependent protein kinase 1 (PDK1), to be highly restricted. A mechanism for FRET restriction based on a highly unfavorable κ2 orientation factor arising from differences in donor-acceptor transition dipole moment angles in a far from coplanar and near static interaction geometry was proposed. Here this is tested via FRET to mC arising from the association of glutathione (GSH) and glutathione S-transferase (GST) with an intrinsically homogeneous and more mobile donor Oregon Green 488 (OG). A new analysis of the acceptor window intensity, based on the turnover point of the sensitized fluorescence, is combined with donor window intensity and anisotropy measurements which show that unrestricted FRET to mC takes place. However, a long-lived anisotropy decay component in the donor window reveals a GST-GSH population in which FRET does not occur, explaining previous discrepancies between quantitative FRET measurements of GST-GSH association and their accepted values. This reinforces the importance of the local donor-acceptor environment in mediating energy transfer and the need to perform spectrally resolved intensity and anisotropy decay measurements in the accurate quantification of fluorescent protein FRET.

Regulation of 3-phosphoinositide-dependent protein kinase 1 activity by homodimerization in live cells.

3-Phosphoinositide-dependent kinase 1 (PDK1) plays a central role in regulating the activity of protein kinases that are essential for signaling; however, how PDK1 itself is regulated is largely unknown. We found that homodimerization of PDK1 is a spatially and temporally regulated mechanism for controlling PDK1 activity. We used Förster resonance energy transfer monitored by fluorescence lifetime imaging microscopy to observe PDK1 homodimerization in live cells. A pleckstrin homology (PH) domain-dependent, basal dimeric association of PDK1 was increased upon cell stimulation with growth factors; this association was prevented by a phosphatidylinositol 3-kinase inhibitor and by a mutation in, or a complete deletion of, the PH domain of PDK1. The distinct spatial distribution of PDK1 homodimers relative to that of heterodimers of PDK1 and protein kinase B (PKB), and the ability of monomeric mutants of PDK1 to phosphorylate PKB, suggested that the monomer was the active conformation. Mutation of the autophosphorylation residue threonine-513 to glutamate, which was predicted to destabilize the homodimer interface, enhanced the interaction between PDK1 and PKB and the activity of PKB. Through in vitro, time-resolved fluorescence intensity and anisotropy measurements, combined with existing crystal structures and computational molecular modeling, we determined the geometrical arrangement of the PDK1 homodimer. With this approach, we calculated the size of the population of PDK1 dimers in cells. This description of a previously uncharacterized regulatory mechanism for the activation of PDK1 offers possibilities for controlling PDK1 activity therapeutically.

Probing nanosecond motions of plasminogen activator inhibitor-1 by time-resolved fluorescence anisotropy.

Plasminogen activator inhibitor-1 (PAI-1) is a member of the serpin (serine protease inhibitor) superfamily. Like most serpins, the inhibitory function of PAI-1 relies on a flexible reactive centre loop (RCL) undertaking a striking conformational transition. We have investigated the conformational dynamics of the RCL of PAI-1 by time-resolved fluorescence anisotropy. A heterogeneous population model with three rotational correlation times has been employed to account for the "dip and rise" observed in fluorescence anisotropy decay curves. The RCL becomes almost fully solvent exposed and exhibits faster rotation when PAI-1 interacts with a RCL-mimicking octapeptide which blocks the loop insertion pathway, indicating that the RCL is well displaced from the protein surface; while the binding of Somatomedin B (SMB) domain of vitronectin, only induces small changes in the RCL. Comparison of the fluorescence lifetime and anisotropy decay of the wild-type PAI-1 with that of the stabilised mutant suggests that there would be no major structural differences between them. Our results indicate that in a native serpin, the P14 residue of the hinge region can flip in and out of the central beta-sheet A more readily than previously thought, which is likely an inherent property for serpins' protease inhibitory function.

Characterization of a single molecule DNA switch in free solution.

We have studied a donor-acceptor fluorophore-labeled DNA switch where the acceptor is Alexa-647, a carbocyanine dye, in solution at the single molecule level to elucidate the fluorescence switching mechanism. The acceptor, which is in an initial high fluorescence trans state, undergoes a photoisomerization reaction resulting in two additional states during its sub-millisecond transit across the probe volume. These two states are assigned to a nonfluorescent triplet trans state that strongly quenches the donor emission and a singlet cis state that blocks the fluorescence resonance energy transfer (FRET) pathway and gives rise to donor-only fluorescence. The formation of these states is faster than the transit time, so that all three states are approximately equally populated under our experimental conditions. The acceptor dye can stick to the DNA in all these states, with the rate of unsticking determining the rate of isomerization into the other states. Measurement of the rate of change of the FRET signal therefore provides information about the fluorophore-DNA intramolecular dynamics. These results explain the large zero peak in the proximity ratio, often seen in single molecule FRET experiments, and suggest that photoinduced effects may be important in single molecule FRET experiments using carbocyanine dyes. They also suggest that for fast photoinduced switching the interactions of the acceptor dye with the DNA and other surfaces should be prevented.

Exit routes from the transition state: Angular momentum constraints on the formation of products.

We have analyzed experimental data from a number of exothermic processes in which molecules in well-defined initial states are deactivated by inelastic, dissociative, or reactive collisions. Further, we analyze deactivation processes that do not occur in molecules despite their containing high levels of excitation. Significant common elements are found among these forms of deactivation. The initial step consists of transition to a product state involving minimum rotation state change (Delta j) consistent with energy conservation. Frequently, this process is near-energy-resonant. More critically, it may frequently require substantial angular momentum (AM) change. Analysis of experimental data indicates that constraints act upon on the formation of products in processes that involve release of excess energy. These constraints are associated with the magnitude of AM that must be generated for the initial transition to occur and this AM "load" increases with the amount of energy to be released. In general, the probability of generating rotational AM falls rapidly as Delta j increases, and this effectively limits the size of energy gap that may be bridged by a given reactant pair and at some point the constraint is sufficient to constitute a barrier that prevents the process from taking place. The choice of reactant species strongly affects the probability of each process that increases (i) when molecules efficiently interconvert momentum and (ii) when many product states are available in the critical near-resonant region. These factors increase the proportion of initial trajectories that possess the energy and momentum necessary to open a "product" channel. Evidence is presented showing that AM load-reduction strategies lead to marked enhancement of rates of collision-induced processes, suggesting that reduction of constraints in the exit channels from the transition state may constitute a previously unrecognized form of catalysis.