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.

Study liquid-liquid phase separation with optical microscopy: A methodology review.

Intracellular liquid-liquid phase separation (LLPS) is a critical process involving the dynamic association of biomolecules and the formation of non-membrane compartments, playing a vital role in regulating biomolecular interactions and organelle functions. A comprehensive understanding of cellular LLPS mechanisms at the molecular level is crucial, as many diseases are linked to LLPS, and insights gained can inform drug/gene delivery processes and aid in the diagnosis and treatment of associated diseases. Over the past few decades, numerous techniques have been employed to investigate the LLPS process. In this review, we concentrate on optical imaging methods applied to LLPS studies. We begin by introducing LLPS and its molecular mechanism, followed by a review of the optical imaging methods and fluorescent probes employed in LLPS research. Furthermore, we discuss potential future imaging tools applicable to the LLPS studies. This review aims to provide a reference for selecting appropriate optical imaging methods for LLPS investigations.

Structural and functional imaging of brains.

Analyzing the complex structures and functions of brain is the key issue to understanding the physiological and pathological processes. Although neuronal morphology and local distribution of neurons/blood vessels in the brain have been known, the subcellular structures of cells remain challenging, especially in the live brain. In addition, the complicated brain functions involve numerous functional molecules, but the concentrations, distributions and interactions of these molecules in the brain are still poorly understood. In this review, frontier techniques available for multiscale structure imaging from organelles to the whole brain are first overviewed, including magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), serial-section electron microscopy (ssEM), light microscopy (LM) and synchrotron-based X-ray microscopy (XRM). Specially, XRM for three-dimensional (3D) imaging of large-scale brain tissue with high resolution and fast imaging speed is highlighted. Additionally, the development of elegant methods for acquisition of brain functions from electrical/chemical signals in the brain is outlined. In particular, the new electrophysiology technologies for neural recordings at the single-neuron level and in the brain are also summarized. We also focus on the construction of electrochemical probes based on dual-recognition strategy and surface/interface chemistry for determination of chemical species in the brain with high selectivity and long-term stability, as well as electrochemophysiological microarray for simultaneously recording of electrochemical and electrophysiological signals in the brain. Moreover, the recent development of brain MRI probes with high contrast-to-noise ratio (CNR) and sensitivity based on hyperpolarized techniques and multi-nuclear chemistry is introduced. Furthermore, multiple optical probes and instruments, especially the optophysiological Raman probes and fiber Raman photometry, for imaging and biosensing in live brain are emphasized. Finally, a brief perspective on existing challenges and further research development is provided.

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.

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.

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.

Improved resolution in fluorescence microscopy with the FRET pairs by time gating.

We investigate the temporal information of nonlinear donor fluorescence in the FRET frustration microscopy. The arrival time of the donor fluorescence varies with the distribution of the excitation laser. The differences of the arrival times between different positions in the excitation spot can be further enhanced in the case of adding a depletion beam upon the FRET probes. The spatial information is encoded in the temporal dynamics of the fluorescent photons from donor molecules and time-gating in detection can be used to increase the spatial resolution.