Recent Advances in Single-Molecule Tracking and Imaging Techniques

Abstract

Since the early 1990s, single-molecule detection in solution at room temperature has enabled direct observation of single biomolecules at work in real time and under physiological conditions, providing insights into complex biological systems that the traditional ensemble methods cannot offer. In particular, recent advances in single-molecule tracking techniques allow researchers to follow individual biomolecules in their native environments for a timescale of seconds to minutes, revealing not only the distinct pathways these biomolecules take for downstream signaling but also their roles in supporting life. In this review, we discuss various single-molecule tracking and imaging techniques developed to date, with an emphasis on advanced three-dimensional (3D) tracking systems that not only achieve ultrahigh spatiotemporal resolution but also provide sufficient working depths suitable for tracking single molecules in 3D tissue models. We then summarize the observables that can be extracted from the trajectory data. Methods to perform single-molecule clustering analysis and future directions are also discussed.

Targeted Single Particle Tracking with Upconverting Nanoparticles

“…Videos were treated using the TrackMate framework, and the obtained tracks were analyzed by a mean square displacement (MSD) analysis. ,, Briefly, MSD values are the means of the squares of the particle displacements at positions separated by a given time lag. MSD-time lag dependence contains information about the particle diffusion coefficient and also allows us to qualitatively estimate the nature of the particle motion, which can be directed (superdiffusive), Brownian, restricted (subdiffusive), or immobile .…”

“…As a result, SPT trajectories with UCNP labels are no longer limited by photophysics but solely by the dynamics of the system and, in particular, the particle displacement outside the field of view and/or focal plane. Even longer trajectories could be anticipated using a feedback loop to keep the particle of interest in the detection volume. , This will provide information on the long-term dynamics of the targeted components, such as, for instance, the accumulation of receptors in particular domains on the plasma membrane. In principle, it should be possible to track the same FcεRI receptor before and after its antigen-induced clustering with other receptors until its recycling and internalization by the cell.…”

“…Single particle tracking (SPT) is a unique microscopy technique that allows for direct visualization of the movement of individual molecules and particles. 1 SPT is based on monitoring photon emitting or scattering labels attached to the targets. If the labels are distributed sufficiently sparsely, they form an image with multiple diffraction-limited spots, from which the labels can be precisely localized with a typical localization precision of 1−30 nm.…”

“…Single particle tracking (SPT) is a unique microscopy technique that allows for direct visualization of the movement of individual molecules and particles . SPT is based on monitoring photon emitting or scattering labels attached to the targets.…”

Centrioles generate two scaffolds with distinct biophysical properties to build mitotic centrosomes

“…To directly test our Spd-2-flux hypothesis in flies, we examined the behaviour of individual fluorescently-tagged Spd-2 molecules in living embryos. Such studies have provided great insight into the behaviour of proteins in their native environment (Nguyen et al, 2023), so we generated transgenic lines expressing Spd-2 fused to a SNAP-tag—a protein that is not fluorescent but which can be covalently coupled to a fluorescent SNAP-ligand (Keppler et al, 2003). We injected the yellow-fluorescent SNAP ligand JF-549 (Grimm et al, 2015) into Drosophila embryos expressing Spd-2-SNAP and Spd-2-NG.…”