Capturing the start point of the virus-cell interaction with high-speed 3D single-virus tracking.

The early stages of the virus-cell interaction have long evaded observation by existing microscopy methods due to the rapid diffusion of virions in the extracellular space and the large three-dimensional cellular structures involved. Here we present an active-feedback single-particle tracking method with simultaneous volumetric imaging of the live cell environment called 3D-TrIm to address this knowledge gap. 3D-TrIm captures the extracellular phase of the infectious cycle in what we believe is unprecedented detail. We report what are, to our knowledge, previously unobserved phenomena in the early stages of the virus-cell interaction, including skimming contact events at the millisecond timescale, orders of magnitude change in diffusion coefficient upon binding and cylindrical and linear diffusion modes along cellular protrusions. Finally, we demonstrate how this method can move single-particle tracking from simple monolayer culture toward more tissue-like conditions by tracking single virions in tightly packed epithelial cells. This multiresolution method presents opportunities for capturing fast, three-dimensional processes in biological systems.

Active-Feedback 3D Single-Molecule Tracking Using a Fast-Responding Galvo Scanning Mirror.

Real-time three-dimensional single-particle tracking (RT-3D-SPT) allows continuous detection of individual freely diffusing objects with high spatiotemporal precision by applying closed-loop active feedback in an optical microscope. However, the current tracking speed in RT-3D-SPT is primarily limited by the response time of the control actuators, impeding long-term observation of fast diffusive objects such as single molecules. Here, we present an RT-3D-SPT system with improved tracking performance by replacing the XY piezoelectric stage with a galvo scanning mirror with an approximately 5 times faster response rate (∼5 kHz). Based on the previously developed 3D single-molecule active real-time tracking (3D-SMART), this new implementation with a fast-responding galvo mirror eliminates the mechanical movement of the sample and allows a more rapid response to particle motion. The improved tracking performance of the galvo mirror-based implementation is verified through simulation and proof-of-principle experiments. Fluorescent nanoparticles and ∼1 kB double-stranded DNA molecules were tracked via both the original piezoelectric stage and new galvo mirror implementations. With the new galvo-based implementation, notable increases in tracking duration, localization precision, and the degree to which the objects are locked to the center of the detection volume were observed. These results suggest that faster control response elements can expand RT-3D-SPT to a broader range of chemical and biological systems.

Growth Kinetics of Single Polymer Particles in Solution via Active-Feedback 3D Tracking.

The ability to directly observe chemical reactions at the single-molecule and single-particle level has enabled the discovery of behaviors otherwise obscured by ensemble averaging in bulk measurements. However powerful, a common restriction of these studies to date has been the absolute requirement to surface tether or otherwise immobilize the chemical reagent/reaction of interest. This constraint arose from a fundamental limitation of conventional microscopy techniques, which could not track molecules or particles rapidly diffusing in three dimensions, as occurs in solution. However, many chemical processes occur entirely in the solution phase, leaving single-particle/-molecule analysis of this critical area of science beyond the scope of available technology. Here, we report the first kinetics studies of freely diffusing and actively growing single polymer-particles at the single-particle level freely diffusing in solution. Active-feedback single-particle tracking was used to capture three-dimensional (3D) trajectories and real-time volumetric images of freely diffusing polymer particles (D ≈ 10-12 m2/s) and extract the growth rates of individual particles in the solution phase. The observed growth rates show that the average growth rate is a poor representation of the true underlying variability in polymer-particle growth behavior. These data revealed statistically significant populations of faster- and slower-growing particles at different depths in the sample, showing emergent heterogeneity while particles are still freely diffusing in solution. These results go against the prevailing premise that chemical processes in freely diffusing solution will exhibit uniform kinetics. We anticipate that these studies will launch new directions of solution-phase, nonensemble-averaged measurements of chemical processes.

Combined online Bayesian and windowed estimation of background and signal localization facilitates active-feedback particle tracking in complex environments.

Despite successes in tracking single molecules in vitro, the extension of active-feedback single-particle methods to tracking rapidly diffusing and unconfined proteins in live cells has not been realized. Since the existing active-feedback localization methods localize particles in real time assuming zero background, they are ill-suited to track in the inhomogeneous background environment of a live cell. Here, we develop a windowed estimation of signal and background levels using recent data to estimate the current particle brightness and background intensity. These estimates facilitate recursive Bayesian position estimation, improving upon current Kalman-based localization methods. Combined, online Bayesian and windowed estimation of background and signal (COBWEBS) surpasses existing 2D localization methods. Simulations demonstrate improved localization accuracy and responsivity in a homogeneous background for selected particle and background intensity combinations. Improved or similar performance of COBWEBS tracking extends to the majority of signal and background combinations explored. Furthermore, improved tracking durations are demonstrated in the presence of heterogeneous backgrounds for multiple particle intensities, diffusive speeds, and background patterns. COBWEBS can accurately track particles in the presence of high and nonuniform backgrounds, including intensity changes of up to three times the particle's intensity, making it a prime candidate for advancing active-feedback single fluorophore tracking to the cellular interior.

Single-nanoparticle electrophoretic mobility determination and trapping using active-feedback 3D tracking.

Nanoparticles (NP) are versatile materials with widespread applications across medicine and engineering. Despite rapid incorporation into drug delivery, therapeutics, and many more areas of research and development, there is a lack of robust characterization methods. Light scattering techniques such as dynamic light scattering (DLS) and electrophoretic light scattering (ELS) use an ensemble-averaged approach to the characterization of nanoparticle size and electrophoretic mobility (EM), leading to inaccuracies when applied to polydisperse or heterogeneous populations. To address this lack of single-nanoparticle characterization, this work applies 3D Single-Molecule Active Real-time Tracking (3D-SMART) to simultaneously determine NP size and EM on a per-particle basis. Single-nanoparticle EM is determined by using active feedback to "lock on" to a single particle and apply an oscillating electric field along one axis. A maximum likelihood approach is applied to extract the single-particle EM from the oscillating nanoparticle position along the field-actuated axis, while mean squared displacement is used along the non-actuated axes to determine size. Unfunctionalized and carboxyl-functionalized polystyrene NPs are found to have unique EM based on their individual size and surface characteristics, and it is demonstrated that single-nanoparticle EM is a more precise tool for distinguishing unique NP preparations than diffusion alone, able to determine the charge number of individual NPs to an uncertainty of less than 30. This method also explored individual nanoparticle EM in various ionic strengths (0.25-5 mM) and found decreased EM as a function of increasing ionic strength, in agreement with results determined via bulk characterization methods. Finally, it is demonstrated that the electric field can be manipulated in real time in response to particle position, resulting in one-dimensional electrokinetic trapping. Critically, this new single-nanoparticle EM determination and trapping method does not require microfluidics, opening the possibility for the exploration of single-nanoparticle EM in live tissue and more comprehensive characterization of nanoparticles in biologically relevant environments.

Hour-Long, Kilohertz Sampling Rate Three-Dimensional Single-Virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions.

A viral infection process covers a large range of spatiotemporal scales. Tracking the viral infection process with fluorescent labels over long durations while maintaining a fast sampling rate requires bright and highly photostable labels. StayGold is a recently identified green fluorescent protein that has a greater photostability and higher signal intensity under identical illumination conditions compared to existing fluorescence protein variants. Here, StayGold protein fusions were used to generate virus-like particles (StayGold-VLPs) to achieve hour-long 3D single-virus tracking (SVT) with 1000 localizations per second (kHz sampling rate) in live cells. The expanded photon budget from StayGold protein fusions prolonged the tracking duration, facilitating a comprehensive study of viral trafficking dynamics with high temporal resolution over long time scales. The development of StayGold-VLPs presents a simple and general VLP labeling strategy for better performance in SVT, enabling exponentially more information to be collected from single trajectories and allowing for the future possibility of observing the entire life cycle of a single virus.

Hour-long, Kilohertz Sampling Rate 3D Single-virus Tracking in Live Cells Enabled by StayGold Fluorescent Protein Fusions.

The viral infection process covers a large range of spatiotemporal scales. Tracking the viral infection process with fluorescent labels over long durations while maintaining a fast sampling rate requires bright and highly photostable labels. StayGold is a recently identified green fluorescent protein that has a greater photostability and higher signal intensity under identical illumination conditions as compared to existing fluorescence protein variants. Here, StayGold protein fusions were used to generate virus-like particles (StayGold-VLPs) to achieve hour-long 3D single-virus tracking (SVT) with one thousand localizations per second (kHz sampling rate) in live cells. The expanded photon budget from StayGold protein fusions prolonged the tracking duration, facilitating a comprehensive study of viral trafficking dynamics with high temporal resolution over long timescales. The development of StayGold-VLPs presents a simple and general VLP labeling strategy for better performance in SVT, enabling exponentially more information to be collected from single trajectories and allowing for the future possibility of observing the whole life cycle of a single virus.

Engineering Lipid Nanoparticles for Enhanced Intracellular Delivery of mRNA through Inhalation.

Despite lipid nanoparticles' (LNPs) success in the effective and safe delivery of mRNA vaccines, an inhalation-based mRNA therapy for lung diseases remains challenging. LNPs tend to disintegrate due to shear stress during aerosolization, leading to ineffective delivery. Therefore, LNPs need to remain stable through the process of nebulization and mucus penetration, yet labile enough for endosomal escape. To meet these opposing needs, we utilized PEG lipid to enhance the surficial stability of LNPs with the inclusion of a cholesterol analog, ß-sitosterol, to improve endosomal escape. Increased PEG concentrations in LNPs enhanced the shear resistance and mucus penetration, while ß-sitosterol provided LNPs with a polyhedral shape, facilitating endosomal escape. The optimized LNPs exhibited a uniform particle distribution, a polyhedral morphology, and a rapid mucosal diffusion with enhanced gene transfection. Inhaled LNPs led to localized protein production in the mouse lung without pulmonary or systemic toxicity. Repeated administration of these LNPs led to sustained protein production in the lungs. Lastly, mRNA encoding the cystic fibrosis transmembrane conductance regulator (CFTR) was delivered after nebulization to a CFTR-deficient animal model, resulting in the pulmonary expression of this therapeutic protein. This study demonstrated the rational design approach for clinical translation of inhalable LNP-based mRNA therapies.