Single Particle Tracking Microscopy Research Articles

Expanding the Palette of SWIR Emitting Nanoparticles Based on Au Nanoclusters for Single-Particle Tracking Microscopy.

Single-molecule localization microscopy has proved promising to unravel the dynamics and molecular architecture of thin biological samples down to nanoscales. For applications in complex, thick biological tissues shifting single-particle emission wavelengths to the shortwave infrared (SWIR also called NIR II) region between 900 to 2100nm, where biological tissues are more transparent is key. To date, mainly single-walled carbon nanotubes (SWCNTs) enable such applications, but they are inherently 1D objects. Here, 0D ultra-small luminescent gold nanoclusters (AuNCs, <3nm) and ≈25nm AuNC-loaded-polymeric particles that can be detected at the single-particle level in the SWIR are presented. Thanks to high brightness and excellent photostability, it is shown that the dynamics of the spherical polymeric particles can be followed at the single-particle level in solution at video rates for minutes. We compared single particle tracking of AuNC-loaded-polymeric particles with that of SWCNT diffusing in agarose gels demonstrating the specificity and complementarity of diffusion properties of these SWIR-emitting nano-objects when exploring a complex environment. This extends the library of photostable SWIR emitting nanomaterials to 0D nano-objects of variable size for single-molecule localization microscopy in the second biological window, opening unprecedented possibilities for mapping the structure and dynamics of complex biological systems.

ExTrack characterizes transition kinetics and diffusion in noisy single-particle tracks.

Single-particle tracking microscopy is a powerful technique to investigate how proteins dynamically interact with their environment in live cells. However, the analysis of tracks is confounded by noisy molecule localization, short tracks, and rapid transitions between different motion states, notably between immobile and diffusive states. Here, we propose a probabilistic method termed ExTrack that uses the full spatio-temporal information of tracks to extract global model parameters, to calculate state probabilities at every time point, to reveal distributions of state durations, and to refine the positions of bound molecules. ExTrack works for a wide range of diffusion coefficients and transition rates, even if experimental data deviate from model assumptions. We demonstrate its capacity by applying it to slowly diffusing and rapidly transitioning bacterial envelope proteins. ExTrack greatly increases the regime of computationally analyzable noisy single-particle tracks. The ExTrack package is available in ImageJ and Python.

Rewriting Textbooks with Single-Particle Tracking Microscopy (Interview with Dr. Robert Tjian)

Rewriting Textbooks with Single-Particle Tracking Microscopy (Interview with Dr. Robert Tjian)

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.

Dopamine D2 Receptors Dimers: How can we Pharmacologically Target Them?

BackgroundDopamine D2 and D3 receptors can form homo- and heterodimers and are important targets in Schizophrenia and Parkinson’s. Recently, many efforts have been made to pharmacologically target these receptor complexes. This review focuses on various strategies to act specifically on dopamine receptor dimers, that are transiently formed.MethodsVarious binding and functional assays were reviewed to study the properties of bivalent ligands, particularly for the dualsteric compound SB269,652. The dimerization of D2 and D3 receptors were analyzed by using single particle tracking microscopy.ResultsThe specific targeting of dopamine D2 and D3 dimers can be achieved with bifunctional ligands, composed of two pharmacophores binding the two orthosteric sites of the dimeric complex. If the target is a homodimer, then the ligand is homobivalent. Instead, if the target is a heterodimer, then the ligand is heterobivalent. However, there is some concern regarding pharmacokinetics and binding properties of such drugs. Recently, a new generation of bitopic compounds with dualsteric properties have been discovered that bind to the orthosteric and the allosteric sites in one monomeric receptor. Regarding dopamine D2 and D3 receptors, a new dualsteric molecule SB269,652 was shown to have selective negative allosteric properties across D2 and D3 homodimers, but it behaves as an orthosteric antagonist on receptor monomer. Targeting dimers is also complicated as they are transiently formed with varying monomer/dimer ratio. Furthermore, this ratio can be altered by administering an agonist or a bifunctional antagonist.ConclusionLast 15 years have witnessed an explosive amount of work aimed at generating bifunctional compounds as a novel strategy to target GPCR homo- and heterodimers, including dopamine receptors. Their clinical use is far from trivial, but, at least, they have been used to validate the existence of receptor dimers in-vitro and in-vivo. The dualsteric compound SB269, 652, with its peculiar pharmacological profile, may offer therapeutic advantages and a better tolerability in comparison with pure antagonists at D2 and D3 receptors and pave the way for a new generation of antipsychotic drugs.

Single particle tracking of internalized metallic nanoparticles reveals heterogeneous directed motion after clathrin dependent endocytosis in mouse chromaffin cells

Most accepted single particle tracking methods are able to obtain high-resolution trajectories for relatively short periods of time. In this work we apply a straightforward combination of single-particle tracking microscopy and metallic nanoparticles internalization on mouse chromaffin cells to unveil the intracellular trafficking mechanism of metallic-nanoparticle-loaded vesicles (MNP-V) complexes after clathrin dependent endocytosis. We found that directed transport is the major route of MNP-Vs intracellular trafficking after stimulation (92.6% of the trajectories measured). We then studied the MNP-V speed at each point along the trajectory, and found that the application of a second depolarization stimulus during the tracking provokes an increase in the percentage of low-speed trajectory points in parallel with a decrease in the number of high-speed trajectory points. This result suggests that stimulation may facilitate the compartmentalization of internalized MNPs in a more restricted location such as was already demonstrated in neuronal and neuroendocrine cells (Bronfman et al 2003 J. Neurosci. 23 3209–20). Although further experiments will be required to address the mechanisms underlying this transport dynamics, our studies provide quantitative evidence of the heterogeneous behavior of vesicles mobility after endocytosis in chromaffin cells highlighting the potential of MNPs as alternative labels in optical microscopy to provide new insights into the vesicles dynamics in a wide variety of cellular environments.

The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery.

Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal degradation appear therefore as highly interdependent phenomena, in such a way that they should be viewed as a single barrier on the route for efficient transfection. As a matter of fact, they should be evaluated in their entirety for the development of optimized non-viral gene delivery vectors.

Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells

While the delivery of small interfering RNAs (siRNAs) is an attractive strategy to treat several clinical conditions, siRNA-nanocarriers’ stability after intravenous administration is still a major obstacle for the development of RNA-interference based therapies. But, although the need for stability is well recognized, the notion that strong stabilization can decrease nanocarriers’ efficiency is sometimes neglected. In this work we evaluated two stealth functionalization strategies to stabilize the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. The nanocarriers were pre- and post-pegylated, forming vectors with different stabilities in biological fluids. The stealth nanocarriers’ behavior was tested under biological mimetic conditions, as the production of stable siRNA-lipoplexes is determinant to achieve efficient intravenous siRNA delivery to cancer cells. Upon incubation in human serum for 2h, by fluorescence Single Particle Tracking microscopy, PEG-coated lipoplexes were found to have better colloidal stability as they could maintain a relatively stable size. In addition, using fluorescence fluctuation spectroscopy, post-pegylation also proved to avoid siRNA dissociation from the nanocarriers in human serum. Concomitantly it was found that PEG-coated lipoplexes improved cellular uptake and transfection efficiency in H1299 cells, and had the ability to silence BCR-ABL, affecting the survival of K562 cells.Based on an efficient cellular internalization, good silencing effect, good siRNA retention and good colloidal stability in human serum, DODAB:MO (2:1) siRNA-lipoplexes coated with PEG-Cer are considered promising nanocarriers for further in vivo validation. Statement of SignificanceThis work describes two stealth functionalization strategies for the stabilization of the previously validated dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) siRNA-lipoplexes. These nanocarriers are capable of efficiently incorporating and delivering siRNA molecules to cells in order to silence genes whose expression is implicated in a pathological condition. The main objective was to functionalize these nanocarriers with a coating conferring protection to siRNA in blood without compromising its efficient delivery to cancer cells, validating the potential of DODAB:MO (2:1) siRNA-lipoplexes as therapeutic vectors. We show that the stealth strategy is determinant to achieve a stable and efficient nanocarrier, and that DODAB:MO mixtures have a very promising potential for systemic siRNA delivery to leukemic cells.

3D dual-virtual-pinhole assisted single particle tracking microscopy

We propose a novel approach for high-speed, three-dimensional single particle tracking (SPT), which we refer to as dual-virtual-pinhole assisted single particle tracking microscopy (DVPaSPTM). DVPaSPTM system can obtain axial information of the sample without optical or mechanical depth scanning, so as to offer numbers of advantages including faster imaging, improved efficiency and a great reduction of photobleaching and phototoxicity. In addition, by the use of the dual-virtual-pinhole, the effect that the quantum yield exerts to the fluorescent signal can be eliminated, which makes the measurement independent of the surroundings and increases the accuracy of the result. DVPaSPTM system measures the intensity within different virtual pinholes of which the radii are given by the host computer. Axial information of fluorophores can be measured by the axial response curve through the ratio of intensity signals. We demonstrated the feasibility of the proposed method by a series of experiments. Results showed that the standard deviation of the axial measurement was 19.2 nm over a 2.5 μm range with 30 ms temporal resolution.

On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids

Cell-derived membrane vesicles that are released in biofluids, like blood or saliva, are emerging as potential non-invasive biomarkers for diseases, such as cancer. Techniques capable of measuring the size and concentration of membrane vesicles directly in biofluids are urgently needed. Fluorescence single particle tracking microscopy has the potential of doing exactly that by labelling the membrane vesicles with a fluorescent label and analysing their Brownian motion in the biofluid. However, an unbound dye in the biofluid can cause high background intensity that strongly biases the fluorescence single particle tracking size and concentration measurements. While such background intensity can be avoided with light sheet illumination, current set-ups require specialty sample holders that are not compatible with high-throughput diagnostics. Here, a microfluidic chip with integrated light sheet illumination is reported, and accurate fluorescence single particle tracking size and concentration measurements of membrane vesicles in cell culture medium and in interstitial fluid collected from primary human breast tumours are demonstrated.

Measuring The Intravitreal Mobility of Nanomedicines with Single-Particle Tracking Microscopy

To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles in the intact vitreous body. Excised bovine eyes were prepared to preserve the fragile structure of the vitreous humor, while permitting high-resolution fluorescence microscopy and single-particle tracking analysis of intravitreally injected nanoparticles. This assay was validated by analyzing polystyrene beads and further employed to evaluate gene nanomedicines composed of poly(amido amine)s and plasmid DNA. The assay was able to distinguish immobilized cationic nanoparticles from mobile PEGylated nanoparticles. PEGylation of the polyplexes resulted in a drastic improvement of their mobility. An ex vivo eye model is presented for studying nanoparticle mobility in intact vitreous humor by single-particle tracking microscopy. These results give important guidelines for developing gene- and drug-delivery nanomedicines that are compatible with intravitreal administration.

Transport of Nanoparticles in Cystic Fibrosis Sputum and Bacterial Biofilms by Single-Particle Tracking Microscopy

The aim of this study was to evaluate the effect of the surface functionalization of model nanoparticles on their mobility in bacterial biofilms and cystic fibrosis sputum. With single-particle tracking microscopy, the mobility of 0.1- and 0.2-µm fluorescent polyethylene glycol (PEG) modified, carboxylate- and N,N-dimethylethylenediamine-modified polystyrene nanospheres were evaluated in fresh cystic fibrosis sputum, as well as Burkholderia multivorans and Pseudomonas aeruginosa biofilms. PEGylation increased the mobility of the particles in sputum and biofilms, while the charged nanospheres were strongly immobilized. However, the transport of the PEGylated nanoparticles was lower in sputum compared with biofilms. Furthermore, the particle transport showed heterogeneity in samples originating from different patients. This study's data suggest that for future nanocarrier design it will be essential to combine PEGylation with a targeting moiety to ensure sufficient mobility in mucus and a better accumulation of the nanoparticles in the biofilm.

Immobilization of Pseudorabies Virus in Porcine Tracheal Respiratory Mucus Revealed by Single Particle Tracking

Pseudorabies virus (PRV) initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated) nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: −31.8±1.5 mV) experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (−49.8±0.6 mV) and positively (36.7±1.1 mV) charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (−9.6±0.8 mV) diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV) was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV was due to complex mucoadhesive interactions including charge interactions rather than size exclusion.

SMAD versus Non-SMAD Signaling Is Determined by Lateral Mobility of Bone Morphogenetic Protein (BMP) Receptors

Bone (or body) morphogenetic proteins (BMPs) belong to the TGFβ superfamily and are crucial for embryonic patterning and organogenesis as well as for adult tissue homeostasis and repair. Activation of BMP receptors by their ligands leads to induction of several signaling cascades. Using fluorescence recovery after photobleaching, FRET, and single particle tracking microscopy, we demonstrate that BMP receptor type I and II (BMPRI and BMPRII) have distinct lateral mobility properties within the plasma membrane, which is mandatory for their involvement in different signaling pathways. Before ligand binding, BMPRI and a subpopulation of BMPRII exhibit confined motion, reflecting preassembled heteromeric receptor complexes. A second free diffusing BMPRII population only becomes restricted after ligand addition. This paper visualizes time-resolved BMP receptor complex formation and demonstrates that the lateral mobility of BMPRI has a major impact in stabilizing heteromeric BMPRI-BMPRII receptor complexes to differentially stimulate SMAD versus non-SMAD signaling.

An ex vivo assay to measure the intravitreal mobility of nanomedicines for retinal gene therapy

AbstractPurpose In retinal gene therapy, intravitreal injection is a promising technique for administering nanomedicines, granted they remain mobile in the vitreous humour. Here, we optimized an ex vivo assay to measure this mobility in intact bovine vitreous. The results from this work and the newly developed methodology are expected to aid in the rational design of nanomedicines for retinal drug delivery.Methods Excised bovine eyes were prepared in such a way to preserve the fragile structure of the vitreous humour, while permitting fluorescence microscopy inside the bovine vitreous. This assay was used to determine by single particle tracking analysis the intravitreal mobility of both model polystyrene beads with different sizes and surface groups, as well as gene nanomedicines composed of poly(amido amine)s and plasmid DNA.Results Cationic nanoparticles were readily immobilized in the vitreous humour, while anionic polystyrene beads remained mobile. Surface modification with the hydrophilic polymer polyethylene glycol (PEG) resulted in the best mobility of the polystyrene beads. In correspondence with these results, the cationic gene nanomedicines were immobilized in the vitreous humour, while modification with PEG resulted in a drastic improvement of their mobility.Conclusion Here an ex vivo assay is presented to study nanoparticle mobility in intact vitreous humour by single particle tracking microscopy, which showed that cationic surface charge limits intravitreal diffusion of nanoparticles by binding to biopolymer structures, while anionic and PEGylated nanoparticles remain mobile. These results should help in the rational design of nanomedicines used for intravitreal drug delivery.

In VivoAnomalous Diffusion and Weak Ergodicity Breaking of Lipid Granules

Combining extensive single particle tracking microscopy data of endogenous lipid granules in living fission yeast cells with analytical results we show evidence for anomalous diffusion and weak ergodicity breaking. Namely we demonstrate that at short times the granules perform subdiffusion according to the laws of continuous time random walk theory. The associated violation of ergodicity leads to a characteristic turnover between two scaling regimes of the time averaged mean squared displacement. At longer times the granule motion is consistent with fractional Brownian motion.

Single particle tracking in systems showing anomalous diffusion: the role of weak ergodicity breaking

Anomalous diffusion has been widely observed by single particle tracking microscopy in complex systems such as biological cells. The resulting time series are usually evaluated in terms of time averages. Often anomalous diffusion is connected with non-ergodic behaviour. In such cases the time averages remain random variables and hence irreproducible. Here we present a detailed analysis of the time averaged mean squared displacement for systems governed by anomalous diffusion, considering both unconfined and restricted (corralled) motion. We discuss the behaviour of the time averaged mean squared displacement for two prominent stochastic processes, namely, continuous time random walks and fractional Brownian motion. We also study the distribution of the time averaged mean squared displacement around its ensemble mean, and show that this distribution preserves typical process characteristics even for short time series. Recently, velocity correlation functions were suggested to distinguish between these processes. We here present analytical expressions for the velocity correlation functions. The knowledge of the results presented here is expected to be relevant for the correct interpretation of single particle trajectory data in complex systems.

Small RNA, Big Impact: Probing miRNA pathways in living cells using single particle tracking

MicroRNAs (miRNAs) are short non‐coding RNAs that control the expression of an estimated 30% of all human proteins post‐transcriptionally. Mature miRNA, typically 21 base pairs long, associates with several proteins to form a miRNA Induced Silencing Complex (miRISC). This complex associates with the Un‐Translated Region (UTR) of mRNAs in a sequence specific manner and forms an active miRISC that controls protein expression. While several different mechanisms have been proposed for miRNA mediated gene repression, they are highly debated. We are investigating the mechanism of miRNA action by observing the diffusive motions of miRISC‐mRNA complexes in living cells using single particle tracking microscopy (SPTM). To this end, we microinject fluorophore labeled miRNA probes into living cultured cells. The probe can bind to multiple sites on its target mRNA which results in an inherent fluorescent amplification and significantly aids in tracking the particle. Quantitative Real Time ‐ Polymerase Chain Reactions (qRT‐PCR) were performed to estimate the level of the HMGA2 mRNA in F9 cells and preliminary tests suggests that there are ~8 mRNA molecules/cell, which is a suitable target number for our SPT experiments. SPT experiments are being performed using which we can deduce whether a specific miRISC‐mRNA complex progresses through multiple mechanisms of repression.Funded by NIGMS.

Particle Tracking Single Protein-Functionalized Quantum Dot Diffusion and Binding at Silica Surfaces

We evaluate commercial QD585 and QD605 streptavidin-functionalized quantum dots (QDs) for single-particle tracking microscopy at surfaces using total internal reflectance fluorescence and measure single QD diffusion and nonspecific binding at silica surfaces in static and flow conditions. The QD diffusion coefficient on smooth, near-ideal, highly hydroxylated silica surfaces is near bulk-solution diffusivity, as expected for repulsive surfaces, but many QD trajectories on rougher, less-than-ideal surfaces or regions display transient adsorptions. We attribute the binding to defect sites or adsorbates, possibly in conjunction with protein conformation changes, and estimate binding energies from the transient adsorption lifetimes. We also assess QD parameters relevant to tracking, including hydrodynamic radius, charge state, signal levels, blinking reduction with reducing solutions, and photoinduced blueing and bleaching.

Analysis and interpretation of two-dimensional single-particle tracking microscopy measurements: Effect of local surface roughness

Methodological advances in light microscopy have made it possible to record the motions of individual lipid and protein molecules resident in the membrane of living cells down to the nanometer level of precision in the x, y plane. Such measurement of a single molecule’s trajectory for a sufficiently long period of time or the measurement of multiple molecules’ trajectories for a shorter period of time can in principle provide the necessary information to derive the particle’s macroscopic two-dimensional-diffusion coefficient—a quantity of vital biological interest. However, one drawback of the light microscopy procedures used in such experiments is their relatively poor discriminatory capability for determining spatial differences along the z axis in comparison to those in the x, y plane. In this study we used computer simulation to examine the likely effect of local surface roughness over the nanometer to micrometer scale on the determination of diffusion constants in the membrane bilayer by the use of such optical-microscope-based single-particle tracking (SPT) procedures. We specifically examined motion of a single molecule along (i) a locally planar and (ii) a locally rough surface. Our results indicate a need for caution in applying overly simplistic analytical strategies to the analysis of data from SPT measurements and provide upper and lower bounds for the likely degree of error introduced on the basis of surface roughness effects alone. Additionally we present an empirical method based on an autocorrelation function approach that may prove useful in identifying the existence of surface roughness and give some idea of its extent.