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.

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.

The trajectory patterns of single HIV-1 virus-like particle in live CD4 cells: A real time three-dimensional multi-resolution microscopy study using encapsulated nonblinking giant quantum dot.

BACKGROUND: The exploration of virology knowledge was limited by the optical technology for the observation of virus. Previously, a three-dimensional multi-resolution real-time microscope system (3D-MRM) was developed to observe the uptake of HIV-1-tat peptide-modified nanoparticles in cell membrane. In this study, we labeled HIV-1 virus-like particles (VLPs) with passivated giant quantum dots (gQDs) and recorded their interactive trajectories with human Jurkat CD4 cells through 3D-MRM. METHODS: The labeled of gQDs of the HIV-1 VLPs in sucrose-gradient purified viral lysates was first confirmed by Cryo-electronic microscopy and Western blot assay. After the infection with CD4 cells, the gQD-labeled VLPs were visualized and their extracellular and intracellular trajectories were recorded by 3D-MRM. RESULTS: A total of 208 prime trajectories was identified and classified into three distinct patterns: cell-free random diffusion pattern, directional movement pattern and cell-associated movement pattern, with distributions and mean durations were 72.6%/87.6 s, 9.1%/402.7 s and 18.3%/68.7 s, respectively. Further analysis of the spatial-temporal relationship between VLP trajectories and CD4 cells revealed the three stages of interactions: (1) cell-associated (extracellular) diffusion stage, (2) cell membrane surfing stage and (3) intracellular directional movement stage. CONCLUSION: A complete trajectory of HIV-1 VLP interacting with CD4 cells was presented in animation. This encapsulating method could increase the accuracy for the observation of HIV-1-CD4 cell interaction in real time and three dimensions.

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.

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.

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.

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.

Particle-by-Particle In†Situ Characterization of the Protein Corona via Real-Time 3D Single-Particle-Tracking Spectroscopy*.

Nanoparticles (NPs) adsorb proteins when exposed to biological fluids, forming a dynamic protein corona that affects their fate in biological environments. A comprehensive understanding of the protein corona is lacking due to the inability of current techniques to precisely measure the full corona in situ at the single-particle level. Herein, we introduce a 3D real-time single-particle tracking spectroscopy to "lock-on" to single freely diffusing polystyrene NPs and probe their individual protein coronas, primarily using bovine serum albumin (BSA) as a model system. The fluorescence signals and diffusive motions of the tracked NPs enable quantification of the "hard corona" using mean-squared displacement analysis. Critically, this method's particle-by-particle nature enabled a lock-in-type frequency filtering approach to extract the full protein corona, despite the typically confounding effect of high background signal from unbound proteins. From these results, the dynamic in situ full protein corona is observed to contain twice the number of proteins compared to the ex situ-measured "hard" protein corona.

Information-Efficient, Off-Center Sampling Results in Improved Precision in 3D Single-Particle Tracking Microscopy.

In this work, we present a 3D single-particle tracking system that can apply tailored sampling patterns to selectively extract photons that yield the most information for particle localization. We demonstrate that off-center sampling at locations predicted by Fisher information utilizes photons most efficiently. When performing localization in a single dimension, optimized off-center sampling patterns gave doubled precision compared to uniform sampling. A ~20% increase in precision compared to uniform sampling can be achieved when a similar off-center pattern is used in 3D localization. Here, we systematically investigated the photon efficiency of different emission patterns in a diffraction-limited system and achieved higher precision than uniform sampling. The ability to maximize information from the limited number of photons demonstrated here is critical for particle tracking applications in biological samples, where photons may be limited.

Author Correction: Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA.

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

Real-time 3D single molecule tracking.

To date, single molecule studies have been reliant on tethering or confinement to achieve long duration and high temporal resolution measurements. Here, we present a 3D single-molecule active real-time tracking method (3D-SMART) which is capable of locking on to single fluorophores in solution for minutes at a time with photon limited temporal resolution. As a demonstration, 3D-SMART is applied to actively track single Atto 647 N fluorophores in 90% glycerol solution with an average duration of ~16 s at count rates of ~10 kHz. Active feedback tracking is further applied to single proteins and nucleic acids, directly measuring the diffusion of various lengths (99 to 1385 bp) of single DNA molecules at rates up to 10 µm2/s. In addition, 3D-SMART is able to quantify the occupancy of single Spinach2 RNA aptamers and capture active transcription on single freely diffusing DNA. 3D-SMART represents a critical step towards the untethering of single molecule spectroscopy.

Mapping solvation heterogeneity in live cells by hyperspectral stimulated Raman scattering microscopy.

Water provides a dynamic matrix in which all biochemical processes occur in living organisms. The structure and dynamics of intracellular water constitute the cornerstone for understanding all aspects of cellular function. Fundamentally, direct visualization of subcellular solvation heterogeneity is essential but remains challenging with commonly used nuclear magnetic resonance methods due to poor spatial resolution. To explore this question, we demonstrate a vibrational-shift imaging approach by combining the spectral-focusing hyperspectral stimulated Raman scattering technique with an environmentally sensitive nitrile probe. The sensing ability of a near-infrared nitrile-containing molecule is validated in the solution phase, microscopic droplets, and cellular environments. Finally, we quantitatively measure the subcellular solvation variance between the cytoplasm (29.5%, S.E. 1.8%) and the nucleus (57.3%, S.E. 1.0%), which is in good agreement with previous studies. This work sheds light on heterogeneous solvation in live systems using coherent Raman microscopy and opens up new avenues to explore environmental variance in complex systems with high spatiotemporal resolution.

Naturally-occurring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA.

Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to delivery. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to -OH group is required to maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit higher cellular uptake and retention, potentially leading to a steady release from the endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape. Our findings show the importance of cholesterol in subcellular transport of LNPs carrying mRNA and emphasize the need for greater insights into surface composition and structural properties of nanoparticles, and their subcellular interactions which enable designs to improve endosomal escape.

Continuous focal translation enhances rate of point-scan volumetric microscopy.

Two-Photon Laser-Scanning Microscopy is a powerful tool for exploring biological structure and function due to its ability to optically section through a sample with a tight focus. While it is possible to obtain 3D image stacks by moving a stage, this per-frame imaging process is time consuming. Here, we present a method for an easy-to-implement and inexpensive modification of an existing two-photon microscope to rapidly image in 3D using an electrically tunable lens to create a tessellating scan pattern which repeats with the volume rate. Using appropriate interpolating algorithms, the volumetric imaging rate can be increased by a factor up to four-fold. This capability provides the expansion of the two-photon microscope into the third dimension for faster volumetric imaging capable of visualizing dynamics on timescales not achievable by traditional stage-stack methods.

An Adaptive Real-Time 3D Single Particle Tracking Method for Monitoring Viral First Contacts.

Here, an adaptive real-time 3D single particle tracking method is proposed, which is capable of capturing heterogeneous dynamics. Using a real-time measurement of a rapidly diffusing particle's positional variance, the 3D precision adaptive real-time tracking (3D-PART) microscope adjusts active-feedback parameters to trade tracking speed for precision on demand. This technique is demonstrated first on immobilized fluorescent nanoparticles, with a greater than twofold increase in the lateral localization precision (≈25 to ≈11 nm at 1 ms sampling) as well as a smaller increase in the axial localization precision (≈ 68 to ≈45 nm). 3D-PART also shows a marked increase in the precision when tracking freely diffusing particles, with lateral precision increasing from ≈100 to ≈70 nm for particles diffusing at 4 µm2 s-1 , although with a sacrifice in the axial precision (≈250 to ≈350 nm). This adaptive microscope is then applied to monitoring the viral first contacts of virus-like particles to the surface of live cells, allowing direct and continuous measurement of the viral particle at initial contact with the cell surface.

Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells.

Single molecule fluorescence spectroscopy has been largely implemented using methods which require tethering of molecules to a substrate in order to make high temporal resolution measurements. However, the act of tethering a molecule requires that the molecule be removed from its environment. This is especially perturbative when measuring biomolecules such as enzymes, which may rely on the non-equilibrium and crowded cellular environment for normal function. A method which may be able to un-tether single molecule fluorescence spectroscopy is real-time 3D single particle tracking (RT-3D-SPT). RT-3D-SPT uses active feedback to effectively lock-on to freely diffusing particles so they can be measured continuously with up to photon-limited temporal resolution over large axial ranges. This review gives an overview of the various active feedback 3D single particle tracking methods, highlighting specialized detection and excitation schemes which enable high-speed real-time tracking. Furthermore, the combination of these active feedback methods with simultaneous live-cell imaging is discussed. Finally, the successes in real-time 3D single molecule tracking (RT-3D-SMT) thus far and the roadmap going forward for this promising family of techniques are discussed.

A Protocol for Real-time 3D Single Particle Tracking.

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 µm2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

Robust real-time 3D single-particle tracking using a dynamically moving laser spot.

Real-time three-dimensional (3D) single-particle tracking uses optical feedback to lock on to freely diffusing nanoscale fluorescent particles, permitting precise 3D localization and continuous spectroscopic interrogation. Here we describe a new method of real-time 3D single-particle tracking wherein a diffraction-limited laser spot is dynamically swept through the detection volume in three dimensions using a two-dimensional (2D) electro-optic deflector and a tunable acoustic gradient lens. This optimized method, called 3D dynamic photon localization tracking (3D-DyPLoT), enables high-speed real-time tracking of single silica-coated non-blinking quantum dots (∼30 nm diameter) with diffusive speeds exceeding 10 µm2/s at count rates as low as 10 kHz, as well as YFP-labeled virus-like particles. The large effective detection area (1 µm×1 µm×4 µm) allows the system to easily pick up fast-moving particles, while still demonstrating high localization precision (σx=6.6 nm, σy=8.7 nm, and σz=15.6 nm). Overall, 3D-DyPLoT provides a fast and robust method for real-time 3D tracking of fast and lowly emitting particles, based on a single excitation and detection pathway, paving the way to more widespread application to relevant biological problems.

Imaging the behavior of molecules in biological systems: breaking the 3D speed barrier with 3D multi-resolution microscopy.

The overwhelming effort in the development of new microscopy methods has been focused on increasing the spatial and temporal resolution in all three dimensions to enable the measurement of the molecular scale phenomena at the heart of biological processes. However, there exists a significant speed barrier to existing 3D imaging methods, which is associated with the overhead required to image large volumes. This overhead can be overcome to provide nearly unlimited temporal precision by simply focusing on a single molecule or particle via real-time 3D single-particle tracking and the newly developed 3D Multi-resolution Microscopy (3D-MM). Here, we investigate the optical and mechanical limits of real-time 3D single-particle tracking in the context of other methods. In particular, we investigate the use of an optical cantilever for position sensitive detection, finding that this method yields system magnifications of over 3000×. We also investigate the ideal PID control parameters and their effect on the power spectrum of simulated trajectories. Taken together, these data suggest that the speed limit in real-time 3D single particle-tracking is a result of slow piezoelectric stage response as opposed to optical sensitivity or PID control.