Search Results (19)

Pulsed focused ultrasound (FUS) in combination with microbubbles has been shown to improve delivery and penetration of nanoparticles in tumors. To understand the mechanisms behind this treatment, it is important to evaluate the contribution of FUS without microbubbles on increased nanoparticle penetration and transport in the tumor extracellular matrix (ECM). A composite agarose hydrogel was made to model the porous structure, the acoustic attenuation and the hydraulic conductivity of the tumor ECM. Single-particle tracking was used as a novel method to monitor nanoparticle dynamics in the hydrogel during FUS exposure. FUS exposure at 1 MHz and 1 MPa was performed to detect any increase in nanoparticle diffusion or particle streaming at acoustic parameters relevant for FUS in combination with microbubbles. Results were compared to a model of acoustic streaming. The nanoparticles displayed anomalous diffusion in the hydrogel, and FUS with a duty cycle of 20% increased the nanoparticle diffusion coefficient by 23%. No increase in diffusion was found for lower duty cycles. FUS displaced the hydrogel itself at duty cycles above 10%; however, acoustic streaming was found to be negligible. In conclusion, pulsed FUS alone cannot explain the enhanced penetration of nanoparticles seen when using FUS and microbubbles for nanoparticle delivery, but it could be used as a tool to enhance diffusion of particles in the tumor ECM. Full article

The function of most lipases is controlled by the lid, which undergoes conformational changes at a water–lipid interface to expose the active site, thus activating catalysis. Understanding how lid mutations affect lipases’ function is important for designing improved variants. Lipases’ function has been found to correlate with their diffusion on the substrate surface. Here, we used single-particle tracking (SPT), a powerful tool for deciphering enzymes’ diffusional behavior, to study Thermomyces lanuginosus lipase (TLL) variants with different lid structures in a laundry-like application condition. Thousands of parallelized recorded trajectories and hidden Markov modeling (HMM) analysis allowed us to extract three interconverting diffusional states and quantify their abundance, microscopic transition rates, and the energy barriers for sampling them. Combining those findings with ensemble measurements, we determined that the overall activity variation in the application condition is dependent on surface binding and lipase mobility when bound. Specifically, the L4 variant with a TLL-like lid and wild-type (WT) TLL displayed similar ensemble activity, but WT bound stronger to the surface than L4, while L4 had a higher diffusion coefficient and thus activity when bound to the surface. These mechanistic elements can only be de-convoluted by our combined assays. Our findings offer fresh perspectives on the development of the next iteration of enzyme-based detergent. Full article

The nanoscale spatiotemporal resolution of single-particle tracking (SPT) renders it a powerful method for exploring single-molecule dynamics in living cells or tissues, despite the disadvantages of using traditional organic fluorescence probes, such as the weak fluorescent signal against the strong cellular autofluorescence background coupled with a fast-photobleaching rate. Quantum dots (QDs), which enable tracking targets in multiple colors, have been proposed as an alternative to traditional organic fluorescence dyes; however, they are not ideally suitable for applying SPT due to their hydrophobicity, cytotoxicity, and blinking problems. This study reports an improved SPT method using silica-coated QD-embedded silica nanoparticles (QD²), which represent brighter fluorescence and are less toxic than single QDs. After treatment of QD² in 10 μg/mL, the label was retained for 96 h with 83.76% of labeling efficiency, without impaired cell function such as angiogenesis. The improved stability of QD² facilitates the visualization of in situ endothelial vessel formation without real-time staining. Cells retain QD² fluorescence signal for 15 days at 4 °C without significant photobleaching, indicating that QD² has overcome the limitations of SPT enabling long-term intracellular tracking. These results proved that QD² could be used for SPT as a substitute for traditional organic fluorophores or single quantum dots, with its photostability, biocompatibility, and superior brightness. Full article

We attempted to examine the alterations elicited by opioids via coexpressed μ-opioid (MOP) and nociceptin/orphanin FQ (NOP) receptors for receptor localization and Erk1/2 (p44/42 MAPK) in human embryonic kidney (HEK) 293 cells. Through two-photon microscopy, the proximity of MOP and NOP receptors was verified by fluorescence resonance energy transfer (FRET), and morphine but not buprenorphine facilitated the process of MOP-NOP heterodimerization. Single-particle tracking (SPT) further revealed that morphine or buprenorphine hindered the movement of the MOP-NOP heterodimers. After exposure to morphine or buprenorphine, receptor localization on lipid rafts was detected by immunocytochemistry, and phosphorylation of Erk1/2 was determined by immunoblotting in HEK 293 cells expressing MOP, NOP, or MOP+NOP receptors. Colocalization of MOP and NOP on lipid rafts was enhanced by morphine but not buprenorphine. Morphine stimulated the phosphorylation of Erk1/2 with a similar potency in HEK 293 cells expressing MOP and MOP+NOP receptors, but buprenorphine appeared to activate Erk1/2 solely through NOP receptors. Our results suggest that opioids can fine-tune the cellular localization of opioid receptors and phosphorylation of Erk1/2 in MOP+NOP-expressing cells. Full article

We recently assisted in a revolution in the realm of fluorescence microscopy triggered by the advent of super-resolution techniques that surpass the classic diffraction limit barrier. By providing optical images with nanometer resolution in the far field, super-resolution microscopy (SRM) is currently accelerating our understanding of the molecular organization of bio-specimens, bridging the gap between cellular observations and molecular structural knowledge, which was previously only accessible using electron microscopy. SRM mainly finds its roots in progress made in the control and manipulation of the optical properties of (single) fluorescent molecules. The flourishing development of novel fluorescent nanostructures has recently opened the possibility of associating super-resolution imaging strategies with nanomaterials’ design and applications. In this review article, we discuss some of the recent developments in the field of super-resolution imaging explicitly based on the use of nanomaterials. As an archetypal class of fluorescent nanomaterial, we mainly focus on single-walled carbon nanotubes (SWCNTs), which are photoluminescent emitters at near-infrared (NIR) wavelengths bearing great interest for biological imaging and for information optical transmission. Whether for fundamental applications in nanomaterial science or in biology, we show how super-resolution techniques can be applied to create nanoscale images “in”, “of” and “with” SWCNTs. Full article

In order to survive, bacterial cells rely on precise spatiotemporal organization and coordination of essential processes such as cell growth, chromosome segregation, and cell division. Given the general lack of organelles, most bacteria are forced to depend on alternative localization mechanisms, such as, for example, geometrical cues. DivIVA proteins are widely distributed in mainly Gram-positive bacteria and were shown to bind the membrane, typically in regions of strong negative curvature, such as the cell poles and division septa. Here, they have been shown to be involved in a multitude of processes: from apical cell growth and chromosome segregation in actinobacteria to sporulation and inhibition of division re-initiation in firmicutes. Structural analyses revealed that DivIVA proteins can form oligomeric assemblies that constitute a scaffold for recruitment of other proteins. However, it remained unclear whether interaction with partner proteins influences DivIVA dynamics. Using structured illumination microscopy (SIM), single-particle tracking (SPT) microscopy, and fluorescent recovery after photobleaching (FRAP) experiments, we show that DivIVA from Corynebacterium glutamicum is mobilized by its binding partner ParB. In contrast, we show that the interaction between Bacillus subtilis DivIVA and its partner protein MinJ reduces DivIVA mobility. Furthermore, we show that the loss of the rod-shape leads to an increase in DivIVA dynamics in both organisms. Taken together, our study reveals the modulation of the polar scaffold protein by protein interactors and cell morphology. We reason that this leads to a very simple, yet robust way for actinobacteria to maintain polar growth and their rod-shape. In B. subtilis, however, the DivIVA protein is tailored towards a more dynamic function that allows quick relocalization from poles to septa upon division. Full article

Single-particle tracking (SPT) has become a powerful tool to quantify transport phenomena in complex media with unprecedented detail. Based on the reconstruction of individual trajectories, a wealth of informative measures become available for each particle, allowing for a detailed comparison with theoretical predictions. While SPT has been used frequently to explore diffusive transport in artificial fluids and inside living cells, intermediate systems, i.e., biochemically active cell extracts, have been studied only sparsely. Extracts derived from the eggs of the clawfrog Xenopus laevis, for example, are known for their ability to support and mimic vital processes of cells, emphasizing the need to explore also the transport phenomena of nano-sized particles in such extracts. Here, we have performed extensive SPT on beads with 20 nm radius in native and chemically treated Xenopus extracts. By analyzing a variety of distinct measures, we show that these beads feature an anti-persistent subdiffusion that is consistent with fractional Brownian motion. Chemical treatments did not grossly alter this finding, suggesting that the high degree of macromolecular crowding in Xenopus extracts equips the fluid with a viscoelastic modulus, hence enforcing particles to perform random walks with a significant anti-persistent memory kernel. Full article

We characterized the size, distribution, and fluidity of microdomains in a lipid bilayer containing phosphatidylinositol (PI) and revealed their roles during the two-dimensional assembly of a membrane deformation protein (FBP17). The morphology of the supported lipid bilayer (SLB) consisting of PI and phosphatidylcholine (PC) on a mica substrate was observed with atomic force microscope (AFM). Single particle tracking (SPT) was performed for the PI+PC-SLB on the mica substrate by using the diagonal illumination setup. The AFM topography showed that PI-derived submicron domains existed in the PI+PC-SLB. The spatiotemporal dependence of the lateral lipid diffusion obtained by SPT showed that the microdomain had lower fluidity than the surrounding region and worked as the obstacles for the lipid diffusion. We observed the two-dimensional assembly of FBP17, which is one of F-BAR family proteins included in endocytosis processes and has the function generating lipid bilayer tubules in vitro. At the initial stage of the FBP17 assembly, the PI-derived microdomain worked as a scaffold for the FBP17 adsorption, and the fluid surrounding region supplied FBP17 to grow the FBP17 domain via the lateral molecular diffusion. This study demonstrated an example clearly revealing the roles of two lipid microregions during the protein reaction on a lipid bilayer. Full article

Single Particle Tracking (SPT) is a powerful class of methods for studying the dynamics of biomolecules inside living cells. The techniques reveal the trajectories of individual particles, with a resolution well below the diffraction limit of light, and from them the parameters defining the motion model, such as diffusion coefficients and confinement lengths. Most existing algorithms assume these parameters are constant throughout an experiment. However, it has been demonstrated that they often vary with time as the tracked particles move through different regions in the cell or as conditions inside the cell change in response to stimuli. In this work, we propose an estimation algorithm to determine time-varying parameters of systems that discretely switch between different linear models of motion with Gaussian noise statistics, covering dynamics such as diffusion, directed motion, and Ornstein–Uhlenbeck dynamics. Our algorithm consists of three stages. In the first stage, we use a sliding window approach, combined with Expectation Maximization (EM) to determine maximum likelihood estimates of the parameters as a function of time. These results are only used to roughly estimate the number of model switches that occur in the data to guide the selection of algorithm parameters in the second stage. In the second stage, we use Change Detection (CD) techniques to identify where the models switch, taking advantage of the off-line nature of the analysis of SPT data to create non-causal algorithms with better precision than a purely causal approach. Finally, we apply EM to each set of data between the change points to determine final parameter estimates. We demonstrate our approach using experimental data generated in the lab under controlled conditions. Full article

This study describes the adaptation of non-linear microscopy for single-particle tracking (SPT), a method commonly used in biology with single-photon fluorescence. Imaging moving objects with non-linear microscopy raises difficulties due to the scanning process of the acquisitions. The interest of the study is based on the balance between all the experimental parameters (objective, resolution, frame rate) which need to be optimized to record long trajectories with the best accuracy and frame rate. To evaluate the performance of the setup for SPT, several basic estimation methods are used and adapted to the new detection process. The covariance-based estimator (CVE) seems to be the best way to evaluate the diffusion coefficient from trajectories using the specific factors of motion blur and localization error. Full article

We investigated lysosome dynamics during neuronal stem cell (NSC) differentiation by two quantitative and complementary biophysical methods based on fluorescence: imaging-derived mean square displacement (iMSD) and single-particle tracking (SPT). The former extracts the average dynamics and size of the whole population of moving lysosomes directly from imaging, with no need to calculate single trajectories; the latter resolves the finest heterogeneities and dynamic features at the single-lysosome level, which are lost in the iMSD analysis. In brief, iMSD analysis reveals that, from a structural point of view, lysosomes decrement in size during NSC differentiation, from 1 μm average diameter in the embryonic cells to approximately 500 nm diameter in the fully differentiated cells. Concomitantly, iMSD analysis highlights modification of key dynamic parameters, such as the average local organelle diffusivity and anomalous coefficient, which may parallel cytoskeleton remodeling during the differentiation process. From average to local, SPT allows mapping heterogeneous dynamic responses of single lysosomes in different districts of the cells. For instance, a dramatic decrease of lysosomal transport in the soma is followed by a rapid increase of transport in the projections at specific time points during neuronal differentiation, an observation compatible with the hypothesis that lysosomal active mobilization shifts from the soma to the newborn projections. Our combined results provide new insight into the lysosome size and dynamics regulation throughout NSC differentiation, supporting new functions proposed for this organelle. Full article

Membrane microdomains are nano-scale domains (10–200 nm) enriched in sterols and sphingolipids. They have many important biological functions, including vesicle transport, endocytosis, and pathogen invasion. A previous study reported that the membrane microdomain-associated protein Flotillin1 (Flot1) was involved in plant development in Arabidopsis thaliana; however, whether sterols affect the plant immunity conveyed by Flot1 is unknown. Here, we showed that the root length in sterol-deficient cyclopropylsterol isomerase 1 (cpi1-1) mutants expressing Flot1 was significantly shorter than in control seedlings. The cotyledon epidermal cells in cpi1-1 mutants expressing Flot1 were smaller than in controls. Moreover, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM) and single-particle tracking (SPT) analysis demonstrated that the long-distance Flot1-GFP movement was decreased significantly in cpi1-1 mutants compared with the control seedlings. Meanwhile, the value of the diffusion coefficient Ĝ was dramatically decreased in cpi1-1 mutants after flagelin22 (flg22) treatment compared with the control seedlings, indicating that sterols affect the lateral mobility of Flot1-GFP within the plasma membrane. Importantly, using confocal microscopy, we determined that the endocytosis of Flot1-GFP was decreased in cpi1-1 mutants, which was confirmed by fluorescence cross spectroscopy (FCS) analysis. Hence, these results demonstrate that sterol composition plays a critical role in the plant defense responses of Flot1. Full article

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. Full article

Lanthanide-doped upconversion nanoparticles (UCNPs) are inorganic nanomaterials in which the lanthanide cations embedded in the host matrix can convert incident near-infrared light to visible or ultraviolet light. These particles are often used for long-term and real-time imaging because they are extremely stable even when subjected to continuous irradiation for a long time. It is now possible to image their movement at the single particle level with a scale of a few nanometers and track their trajectories as a function of time with a scale of a few microseconds. Such UCNP-based single-particle tracking (SPT) technology provides information about the intracellular structures and dynamics in living cells. Thus far, most imaging techniques have been built on fluorescence microscopic techniques (epifluorescence, total internal reflection, etc.). However, two-dimensional (2D) images obtained using these techniques are limited in only being able to visualize those on the focal planes of the objective lens. On the contrary, if three-dimensional (3D) structures and dynamics are known, deeper insights into the biology of the thick cells and tissues can be obtained. In this review, we introduce the status of the fluorescence imaging techniques, discuss the mathematical description of SPT, and outline the past few studies using UCNPs as imaging probes or biologically functionalized carriers. Full article

Single-particle tracking (SPT) has been used and developed over the last 25 years as a method to investigate molecular dynamics, structure, interactions, and function in the cellular context. SPT is able to show how fast and how far individual molecules move, identify different dynamic populations, measure the duration and strength of intermolecular interactions, and map out structures on the nanoscale in cells. In combination with other techniques such as macromolecular crystallography and molecular dynamics simulation, it allows us to build models of complex structures, and develop and test hypotheses of how these complexes perform their biological roles in health as well as in disease states. Here, we use the example of the epidermal growth factor receptor (EGFR), which has been studied extensively by SPT, demonstrating how the method has been used to increase our understanding of the receptor’s organization and function, including its interaction with the plasma membrane, its activation, clustering, and oligomerization, and the role of other receptors and endocytosis. The examples shown demonstrate how SPT might be employed in the investigation of other biomolecules and systems. Full article