RESUMO
We propose to capture reaction-diffusion on a molecule-by-molecule basis from the fastest acquirable timescale, namely individual photon arrivals. We illustrate our method on intrinsically disordered human proteins, the linker histone H1.0 as well as its chaperone prothymosin α, as these diffuse through an illuminated confocal spot and interact forming larger ternary complexes on millisecond timescales. Most importantly, single-molecule reaction-diffusion, smRD, reveals single molecule properties without trapping or otherwise confining molecules to surfaces. We achieve smRD within a Bayesian paradigm and term our method Bayes-smRD. Bayes-smRD is further free of the average, bulk, results inherent to the analysis of long photon arrival traces by fluorescence correlation spectroscopy. In learning from thousands of photon arrivals continuous spatial positions and discrete conformational and photophysical state changes, Bayes-smRD estimates kinetic parameters on a molecule-by-molecule basis with two to three orders of magnitude less data than tools such as fluorescence correlation spectroscopy thereby also dramatically reducing sample photodamage.
RESUMO
Bdellovibrio bacteriovorus is a predatory bacterium preying upon Gram-negative bacteria. As such, B. bacteriovorus has the potential to control antibiotic-resistant pathogens and biofilm populations. To survive and reproduce, B. bacteriovorus must locate and infect a host cell. However, in the temporary absence of prey, it is largely unknown how B. bacteriovorus modulate their motility patterns in response to physical or chemical environmental cues to optimize their energy expenditure. To investigate B. bacteriovorus' predation strategy, we track and quantify their motion by measuring speed distributions as a function of starvation time. While an initial unimodal speed distribution relaxing to one for pure diffusion at long times may be expected, instead we observe a bimodal speed distribution with one mode centered around that expected from diffusion and the other centered at higher speeds. What is more, for an increasing amount of time over which B. bacteriovorus is starved, we observe a progressive reweighting from the active swimming state to an apparent diffusive state in the speed distribution. Distributions of trajectory-averaged speeds for B. bacteriovorus are largely unimodal, indicating switching between a faster swim speed and an apparent diffusive state within individual observed trajectories rather than there being distinct active swimming and apparent diffusive populations. We also find that B. bacteriovorus' apparent diffusive state is not merely caused by the diffusion of inviable bacteria as subsequent spiking experiments show that bacteria can be resuscitated and bimodality restored. Indeed, starved B. bacteriovorus may modulate the frequency and duration of active swimming as a means of balancing energy consumption and procurement. Our results thus point to a reweighting of the swimming frequency on a trajectory basis rather than a population level basis.
Assuntos
Bdellovibrio bacteriovorus , Natação , Sinais (Psicologia) , Bdellovibrio bacteriovorus/fisiologia , Bactérias , BiofilmesRESUMO
Assessing dynamic processes at single molecule scales is key toward capturing life at the level of its molecular actors. Widefield superresolution methods, such as STORM, PALM, and PAINT, provide nanoscale localization accuracy, even when distances between fluorescently labeled single molecules ("emitters") fall below light's diffraction limit. However, as these superresolution methods rely on rare photophysical events to distinguish emitters from both each other and background, they are largely limited to static samples. In contrast, here we leverage spatiotemporal correlations of dynamic widefield imaging data to extend superresolution to simultaneous multiple emitter tracking without relying on photodynamics even as emitter distances from one another fall below the diffraction limit. We simultaneously determine emitter numbers and their tracks (localization and linking) with the same localization accuracy per frame as widefield superresolution does for immobilized emitters under similar imaging conditions (≈50nm). We demonstrate our results for both in cellulo data and, for benchmarking purposes, on synthetic data. To this end, we avoid the existing tracking paradigm relying on completely or partially separating the tasks of emitter number determination, localization of each emitter, and linking emitter positions across frames. Instead, we develop a fully joint posterior distribution over the quantities of interest, including emitter tracks and their total, otherwise unknown, number within the Bayesian nonparametric paradigm. Our posterior quantifies the full uncertainty over emitter numbers and their associated tracks propagated from origins including shot noise and camera artefacts, pixelation, stochastic background, and out-of-focus motion. Finally, it remains accurate in more crowded regimes where alternative tracking tools cannot be applied.
RESUMO
Tracking single molecules continues to provide new insights into the fundamental rules governing biological function. Despite continued technical advances in fluorescent and non-fluorescent labeling as well as data analysis, direct observations of trajectories and interactions of multiple molecules in dense environments remain aspirational goals. While confocal methods provide a means to deduce dynamical parameters with high temporal resolution, such as diffusion coefficients, they do so at the expense of spatial resolution. Indeed, on account of a confocal volume's symmetry, typically only distances from the center of the confocal spot can be deduced. Motivated by the need for true three dimensional high speed tracking in densely labeled environments, we propose a computational tool for tracking many fluorescent molecules traversing multiple, closely spaced, confocal measurement volumes providing independent observations. Various realizations of this multiple confocal volumes strategy have previously been used for long term, large area, tracking of one fluorescent molecule in three dimensions. What is more, we achieve tracking by directly using single photon arrival times to inform our likelihood and exploit Hamiltonian Monte Carlo to efficiently sample trajectories from our posterior within a Bayesian nonparametric paradigm. A nonparametric paradigm here is warranted as the number of molecules present are, themselves, a priori unknown. Taken together, we provide a computational framework to infer trajectories of multiple molecules at once, below the diffraction limit (the width of a confocal spot), in three dimensions at sub-millisecond or faster time scales.