Single Molecule Tracking Techniques Research Articles

Recent Advances in Single-Molecule Tracking and Imaging Techniques.

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.

Impact of Saccharomyces cerevisiae on the Field of Single-Molecule Biophysics.

Cellular functions depend on the dynamic assembly of protein regulator complexes at specific cellular locations. Single Molecule Tracking (SMT) is a method of choice for the biochemical characterization of protein dynamics in vitro and in vivo. SMT follows individual molecules in live cells and provides direct information about their behavior. SMT was successfully applied to mammalian models. However, mammalian cells provide a complex environment where protein mobility depends on numerous factors that are difficult to control experimentally. Therefore, yeast cells, which are unicellular and well-studied with a small and completely sequenced genome, provide an attractive alternative for SMT. The simplicity of organization, ease of genetic manipulation, and tolerance to gene fusions all make yeast a great model for quantifying the kinetics of major enzymes, membrane proteins, and nuclear and cellular bodies. However, very few researchers apply SMT techniques to yeast. Our goal is to promote SMT in yeast to a wider research community. Our review serves a dual purpose. We explain how SMT is conducted in yeast cells, and we discuss the latest insights from yeast SMT while putting them in perspective with SMT of higher eukaryotes.

Impact of Chain Conformation on Structural Heterogeneity in Polymer Network.

Polymer networks generally consist of an ensemble of single chains. However, understanding how chain conformation affects the structure and properties of polymer networks remains a challenge for optimizing their functionality. Here, we present the fabrication and comparative study of a polymer network composed of collapsed self-entangled chains (intrachain entangled network) and a standard polymer network in which random-coil chains are entangled with each other (interchain entangled network). For poly(methyl methacrylate) thin films composed of these networks, we coupled solvent vapor swelling and single-molecule tracking techniques to examine the anomalies in the dynamics of a small-molecular probe included in the system. We demonstrate that when compared to the interchain entangled network the intrachain one exhibits a more substantial structural heterogeneity, particularly under highly crowded conditions. This network also exhibits physical compactness, which keeps the heterogeneous network structure frozen over time and impedes network plasticization through solvent uptake by the film.

Direct measurements of mRNA translation kinetics in living cells

Ribosome mediated mRNA translation is central to life. The cycle of translation, however, has been characterized mostly using reconstituted systems, with only few techniques applicable for studies in the living cell. Here we describe a live-cell ribosome-labeling method, which allows us to characterize the whole processes of finding and translating an mRNA, using single-molecule tracking techniques. We find that more than 90% of both bacterial ribosomal subunits are engaged in translation at any particular time, and that the 30S and 50S ribosomal subunits spend the same average time bound to an mRNA, revealing that 30S re-initiation on poly-cistronic mRNAs is not prevalent in E. coli. Instead, our results are best explained by substantial 70S re-initiation of translation of poly-cistronic mRNAs, which is further corroborated by experiments with translation initiation inhibitors. Finally, we find that a variety of previously described orthogonal ribosomes, with altered anti-Shine-Dalgarno sequences, show significant binding to endogenous mRNAs.

Bridging the gap between single molecule and bulk fluorescence techniques with PIE-FCCS

G protein-coupled receptors (GPCRs) are a diverse set of transmembrane proteins with large variations in sequence and domain topology. One important aspect of GPCR function is the role of spatial organization and dimerization/oligomerization, but there is still no systematic understanding of these effects. This is due, in part, to the techniques that have been used to analyze them. Bulk fluorescence techniques cannot fully quantify receptor concentration, diffusion and oligomerization state. Single molecule tracking techniques are excellent at determining membrane protein diffusion and protein-protein interactions, but the technology requires very low expression levels and so is challenging to perform at physiological expression levels.

Single-molecule imaging of epigenetic complexes in living cells: insights from studies on Polycomb group proteins.

Chromatin-associated factors must locate, bind to, and assemble on specific chromatin regions to execute chromatin-templated functions. These dynamic processes are essential for understanding how chromatin achieves regulation, but direct quantification in living mammalian cells remains challenging. Over the last few years, live-cell single-molecule tracking (SMT) has emerged as a new way to observe trajectories of individual chromatin-associated factors in living mammalian cells, providing new perspectives on chromatin-templated activities. Here, we discuss the relative merits of live-cell SMT techniques currently in use. We provide new insights into how Polycomb group (PcG) proteins, master regulators of development and cell differentiation, decipher genetic and epigenetic information to achieve binding stability and highlight that Polycomb condensates facilitate target-search efficiency. We provide perspectives on liquid-liquid phase separation in organizing Polycomb targets. We suggest that epigenetic complexes integrate genetic and epigenetic information for target binding and localization and achieve target-search efficiency through nuclear organization.

Diffusion behavior of peptide amphiphiles containing different numbers of alkyl tails at a hydrophobic solid-liquid interface: single molecule tracking investigation.

Using the single molecule tracking technique, the diffusion behavior of peptide amphiphiles (PAs) with different numbers of alkyl tails at a hydrophobic solid-liquid interface has been investigated. The effect of the number of alkyl tails of PAs on molecular trajectories at the hydrophobic solid-liquid interface has been systematically studied. PA molecules display an intermittent motion consisting of immobilization and hopping processes, which has been well simulated by the continuous time random walk (CTRW) model. The results reveal that the hydrophobic interaction between the PAs and hydrophobic surface plays an important role in the diffusion behavior of PAs. Increasing the number of alkyl tails in PAs systematically reduces the mobility of PAs on the hydrophobic surface. Moreover, the diffusion behavior of PAs at the hydrophobic interface also shows pH dependence. A decrease in pH is beneficial to the motion of all PAs on the hydrophobic surface, which can be ascribed to the protonation of PAs in acidic solutions. Therefore, the hydrophobic interaction is crucial to the transport of peptide amphiphiles at hydrophobic interfaces which would be important for the design of peptides in biological applications.

Dynamics of mRNA entry into stress granules.

Stressed eukaryotic cells store mRNAs in protein-rich condensates called stress granules. Using single-molecule tracking techniques to examine how mRNAs enter stress granules, a new study shows that mRNAs make transient contacts with the granule surface before stable association, and become largely immobile after entry.

The electric double layer structure modulates poly-dT25 conformation and adsorption kinetics at the cationic lipid bilayer interface.

The conformation and adsorption kinetics of oligonucleotides at lipid membrane interfaces are crucial to their biological functions, but are yet not clearly understood. Poly-dT oligonucleotide molecules have been widely used as primers for reverse translation of RNA molecules, as well as a surface recognition agent for mRNA purification and extraction. In this research, the adsorption processes of poly-dT25 on lipid membranes in different ionic solutions were investigated by sum frequency generation vibrational spectroscopy (SFG-VS) together with a single molecule tracking technique in situ and in real time. These systematic studies provide us with molecular insight into the chemical and physical nature of oligonucleotide-membrane interactions, and show us how the electric double layer (EDL) structure changes the conformation and adsorption kinetics of oligonucleotides. The SFG-VS results indicate that an increase of ionic concentration not only decreases the adsorption density of oligonucleotides but also changes the conformation of oligonucleotides from an elongated conformation to a coiled conformation, causing stronger thermodynamic interactions with membranes, as demonstrated by single molecule tracking techniques. It is also shown that the ionic solution can tune the balance between the surface diffusion rate and solution diffusion rate of oligonucleotides significantly. These results demonstrated that the spectra and kinetics collected by in situ label-free SFG-VS detection and the single molecular tracking technique can provide new molecular insights into the mechanisms of oligonucleotide-membrane interactions. These new understandings may help researchers to control the assembly of oligonucleotide-liposome complexes and to improve the efficiency of transportation and delivery of oligonucleotide molecules.

Measuring Oligonucleotide Hybridization Kinetics in Solution using a time-Resolved 3D Single-Molecule Tracking Technique

Single-molecule measurements of DNA hybridization kinetics are mostly performed on a surface or inside a trap. Here we demonstrate a time-resolved, 3D single-molecule tracking (3D-SMT) method that allows us to follow a freely diffusing ssDNA molecule in solution for hundreds of milliseconds or even seconds and observe multiple annealing and melting events taking place on the same molecule. This is achieved by combining confocal-feedback 3D-SMT with time-domain fluorescence lifetime measurement, where fluorescence lifetime serves as the indicator of hybridization. With sub-diffraction-limit spatial resolution in molecular tracking and 15 ms temporal resolution in monitoring the change of reporter's lifetime, we have demonstrated a full characterization of annealing rate (kon = 5.13 × 106 M−1 s−1), melting rate (koff = 9.55 s−1), and association constant (Ka = 0.54 μM−1) of an 8 bp duplex model system diffusing at 4.8 μm2 s−1. As our method completely eliminates the photobleaching artifacts and diffusion interference, our kon and koff results well represent the real kinetics in solution. Our binding kinetics measurement can be carried out in a low signal-to-noise ratio condition (SNR ≈ 1.4) where ∼130 recorded photons are sufficient for a lifetime estimation. Using a population-level analysis, we can characterize hybridization kinetics over a wide range (0.5-125 s−1), even beyond the reciprocals of the lifetime monitoring temporal resolution and the average track duration. To apply this technique to study hybridization kinetics in live cells, we are testing various oxygen scavenger systems that are live-cell compatible. Our preliminary results have shown improved dye stability in our tracking experiments.

A mobile precursor determines protein resistance on nanostructured surfaces.

Biomaterials are often engineered with nanostructured surfaces to control interactions with proteins and thus regulate their biofunctions. However, the mechanism of how nanostructured surfaces resist or attract proteins together with the underlying design rules remains poorly understood at a molecular level, greatly limiting attempts to develop high-performance biomaterials and devices through the rational design of nanostructures. Here, we study the dynamics of nonspecific protein adsorption on block copolymer nanostructures of varying adhesive domain areas in a resistant matrix. Using surface plasmon resonance and single molecule tracking techniques, we show that weakly adsorbed proteins with two-dimensional diffusivity are critical precursors to protein resistance on nanostructured surfaces. The adhesive domain areas must be more than tens or hundreds of times those of the protein footprints to slow down the 2D-mobility of the precursor proteins for their irreversible adsorption. This precursor model can be used to quantitatively analyze the kinetics of nonspecific protein adsorption on nanostructured surfaces. Our method is applicable to precisely manipulate protein adsorption and resistance on various nanostructured surfaces, e.g., amphiphilic, low-surface-energy, and charged nanostructures, for the design of protein-compatible materials.

Multicolor 3D Single Particle Tracking using Spectrally Displaced Localization

Single particle tracking (SPT) techniques such as sptPALM, uPAINT, and quantum dot tracking have given unprecedented insight into molecular dynamics in living cells. They allow monitoring the behavior of individual proteins in the plasma membrane as well as their molecular interaction with scaffold proteins at millisecond temporal resolution and high spatial resolution (<30 nm) by fitting the point spread function (PSF) of the individual emitter. While these SPT methods have been extended to study the temporal dynamics and co-organization of multiple proteins, conventional experimental setups used to perform multicolor imaging are typically limited to two simultaneous wavelengths. Increasing the number of colors requires additional filters for specific fluorescent tags and is usually performed at the expense of a loss of spatial or temporal resolution. This limits the minimum diffusion coefficient that can be measured, thereby degrading the ability to differentiate between molecular diffusion regimes like immobilization and confined diffusion.By employing a dual collection objective imaging configuration compatible with live cell imaging, we will present a single molecule tracking technique that allows for simultaneous 3D single particle tracking of multiple distinct species without compromising the spatio-temporal resolution. A dispersive element introduced into the second optical path induces a spectrally-dependent displacement, which is used to separate numerous fluorescent species of single emitters based on their emission spectra. We will present the system's spectral separation capabilities in a quantitative manner and validate the technique in fixed samples. We will conclude by demonstrating the ability to simultaneously track several fluorescing species in live cells.

Nanoparticles in Bioimaging

This Special Issue of Nanomaterials is dedicated to the application of nanoparticulate materials in biological imaging.[...].

Single-Molecule Tracking and Its Application in Biomolecular Binding Detection.

In the past two decades significant advances have been made in single-molecule detection, which enables the direct observation of single biomolecules at work in real time and under physiological conditions. In particular, the development of single-molecule tracking (SMT) microscopy allows us to monitor the motion paths of individual biomolecules in living systems, unveiling the localization dynamics and transport modalities of the biomolecules that support the development of life. Beyond the capabilities of traditional camera-based tracking techniques, state-of-the-art SMT microscopies developed in recent years can record fluorescence lifetime while tracking a single molecule in the 3D space. This multiparameter detection capability can open the door to a wide range of investigations at the cellular or tissue level, including identification of molecular interaction hotspots and characterization of association/dissociation kinetics between molecules. In this review, we discuss various SMT techniques developed to date, with an emphasis on our recent development of the next generation 3D tracking system that not only achieves ultrahigh spatiotemporal resolution but also provides sufficient working depth suitable for live animal imaging. We also discuss the challenges that current SMT techniques are facing and the potential strategies to tackle those challenges.

A stochastic spatio-temporal (SST) model to study cell-to-cell variability in HIV-1 infection

Although HIV viremia in infected patients proceeds in a manner that may be accounted for by deterministic mathematical models, single virus-cell encounters following initial HIV exposure result in a variety of outcomes, only one of which results in a productive infection. The development of single molecule tracking techniques in living cells allows studies of intracellular transport of HIV, but it remains less clear what its impact may be on viral integration efficiency. Here, we present a stochastic intracellular mathematical model of HIV replication that incorporates microtubule transport of viral components. Using this model, we could study single round infections and observe how viruses entering cells reach one of three potential fates – degradation of the viral RNA genome, formation of LTR circles, or successful integration and establishment of a provirus. Our model predicts global trafficking properties, such as the probability and the mean time for a HIV viral particle to reach the nuclear pore. Interestingly, our model predicts that trafficking determines neither the probability or time of provirus establishment – instead, they are a function of vRNA degradation and reverse transcription reactions. Thus, our spatio-temporal model provides novel insights into the HIV infection process and may constitute a useful tool for the identification of promising drug targets.

Single-Molecule Imaging Reveals Topology Dependent Mutual Relaxation of Polymer Chains

The motion and relaxation of linear and cyclic polymers under entangled conditions are investigated by means of a newly developed single-molecule tracking technique, cumulative-area (CA) tracking. CA tracking enables simultaneous quantitative characterization of the diffusion mode, diffusion rate, and relaxation time that have been impossible with a widely used conventional single-molecule localization and tracking method, by analyzing cumulative areas occupied by the moving molecule. Using the novel approach, we investigate the motion and relaxation of entangled cyclic polymers, which have been an important but poorly understood question. Fluorescently labeled 42 kbp linear or cyclic tracer dsDNAs in concentrated solutions of unlabeled linear or cyclic DNAs are used as model systems. We show that CA tracking can explicitly distinguish topology-dependent diffusion mode, rate, and relaxation time, demonstrating that the method provides an invaluable tool for characterizing topological interaction between t...

Real time ligand-induced motion mappings of AChBP and nAChR using X-ray single molecule tracking.

We observed the dynamic three-dimensional (3D) single molecule behaviour of acetylcholine-binding protein (AChBP) and nicotinic acetylcholine receptor (nAChR) using a single molecule tracking technique, diffracted X-ray tracking (DXT) with atomic scale and 100 μs time resolution. We found that the combined tilting and twisting motions of the proteins were enhanced upon acetylcholine (ACh) binding. We present the internal motion maps of AChBP and nAChR in the presence of either ACh or α-bungarotoxin (αBtx), with views from two rotational axes. Our findings indicate that specific motion patterns represented as biaxial angular motion maps are associated with channel function in real time and on an atomic scale.

Shape matters in protein mobility within membranes

The lateral mobility of proteins within cell membranes is usually thought to be dependent on their size and modulated by local heterogeneities of the membrane. Experiments using single-particle tracking on reconstituted membranes demonstrate that protein diffusion is significantly influenced by the interplay of membrane curvature, membrane tension, and protein shape. We find that the curvature-coupled voltage-gated potassium channel (KvAP) undergoes a significant increase in protein mobility under tension, whereas the mobility of the curvature-neutral water channel aquaporin 0 (AQP0) is insensitive to it. Such observations are well explained in terms of an effective friction coefficient of the protein induced by the local membrane deformation.

Roles of Cytoskeletal Components on the Membrane Movement of Individual Bk Ca Channels in Live COS-7 cell

Membrane dynamics of the large-conductance Ca2+-activated K+ channels (BKCa channels) was investigated in COS-7 cells by co-expressing the α subunit of the channel tagged with an acceptor peptide for biotin at its extracellular N-terminus and a genetically modified E. coli biotin ligase. We were able to visualize individual BKCa channels that had been biotinylated metabolically and then expressed on to the surface of the cells using quantum dots (QDs) coated with streptavidin. The time-lapse imaging on hundreds of homomeric BKCa channel revealed that the channels in live COS-7 cells exhibited a ‘confined diffusion’ in an area of 1.915 μm2 with an initial diffusion coefficient of 0.033 μm2/s. In order to understand the roles of cytoskeleton on the diffusional characteristics of the channel, we monitored the channel movement in the presence of drugs known to disrupt the filamentous actin and the microtubule, and quantified their effects on channel dynamics. It was found that both diffusion coefficient and the area of diffusion are differentially affected by such drug treatments. We are currently investigating the domain(s) of the channel responsible for cytoskeletal influence on the membrane dynamics of BKCa channel. Using this QD-based, single-molecule tracking technique, we wish to understand the cellular players influencing the diffusional movement of BKCa channel and their mechanism of action. ACKNOWLEDGEMENTS The authors are grateful to the members of the Laboratory of Molecular Neurobiology at GIST for their valuable comments and the Bio-Imaging Research Center at GIST for providing various imaging equipments. This research was supported by the grants from the Bio-Imaging Research Center to C-S Park.

Movements of Individual BK Ca Channels in Live Cell Membrane Monitored by Site-Specific Labeling Using Quantum Dots

The movements of BK Ca channels were investigated in live cells using quantum dots (QDs). The extracellular N-terminus was metabolically tagged with biotin, labeled with streptavidin-conjugated QDs and then monitored using real-time time-lapse imaging in COS-7 cells and cultured neurons. By tracking hundreds of channels, we were able to determine the characteristics of channel movements quantitatively. Channels in COS-7 cells exhibited a confined diffusion in an area of 1.915 μm 2, with an initial diffusion coefficient of 0.033 μm 2/s. In neurons, the channel movements were more heterogeneous and highly dependent on subcellular location. While the channels in soma diffused slowly without clear confinement, axodendritic channels showed more rapid and pseudo-one-dimensional movements. Intriguingly, the channel movement in somata was drastically increased by the neuronal β4 subunit, in contrast to the channels in the axodendritic area where the mobility were significantly decreased. Thus, our results demonstrate that the membrane mobility of BK Ca channels can be greatly influenced by the expression system used, subunit composition, and subcellular location. This QD-based, single-molecule tracking technique can be utilized to investigate the cellular mechanisms that determine the mobility as well as the localization of various membrane proteins in live cells.