Interferometric Scattering Microscopy Research Articles

Long-Range Three-Dimensional Tracking of Nanoparticles Using Interferometric Scattering Microscopy.

Tracking nanoparticle movement is highly desirable in many scientific areas, and various imaging methods have been employed to achieve this goal. Interferometric scattering (iSCAT) microscopy has been particularly successful in combining very high spatial and temporal resolution for tracking small nanoparticles in all three dimensions. However, previous works have been limited to an axial range of only a few hundred nanometers. Here, we present a robust and efficient measurement and analysis strategy for three-dimensional tracking of nanoparticles at high speed and with nanometer precision. After discussing the principle of our approach using synthetic data, we showcase the performance of the method by tracking gold nanoparticles with diameters ranging from 10 to 80 nm in water, demonstrating an axial tracking range from 4 μm for the smallest particles up to over 30 μm for the larger ones. We point out the limitations and robustness of our system across various noise levels and discuss its promise for applications in cell biology and material science, where the three-dimensional motion of nanoparticles in complex media is of interest.

Hybrid interfacial cryosoret nano-engineering in photonic resonator interferometric scattering microscopy: Insights from nanoparticles and nano-assemblies

The requirements of augmented signal contrast provided by nanoparticle tags in biosensor microscopy-based point-of-care technologies for cancer and infectious disease diagnostics can be addressed through metallo-dielectric nanoarchitectures that enhance optical scattering and absorption to provide digital resolution detection of single tags with simple instrumentation. Photonic Resonator Interferometric Scattering Microscopy (PRISM) enables label-free visualization of nanometer-scale analytes such as extracellular vesicles and virions, and its applicability can be extended to biomolecular analyte counting through nanoparticle tags. Here, we present template-free, linker-less cryosoret nano-assemblies fabricated via adiabatic cooling (−196 °C) as plasmonic nano-antennas that provide high scattering contrast in PRISM. Plasmonic Ag and Au nanomaterials and their cryosorets are evaluated through imaging experiments and simulations based on the finite element method to understand the photo-plasmonic coupling effect at the surface of a photonic crystal (PC) interface. The Ag and Au cryosorets provide at most 8.29-fold and 6.77-fold higher signal contrast compared to their singlet counterpart. Through the simulations, the averaged field magnitude enhancements of 2.77-fold and 3.68-fold are observed for Ag and Au cryosorets when interfacing with PCs compared to bare glass substrates. The hybrid coupling between the localized Mie and delocalized Bragg plasmons of cryosorets and the underlying PC's guided mode resonance provides insights for developing nano-assembly-based nano-tags for biosensing applications.

Lanthanide Ion Resonance‐Driven Rayleigh Scattering of Nanoparticles for Dual‐Modality Interferometric Scattering Microscopy (Adv. Sci. 32/2022)

Interferometric Scattering Microscopy Rayleigh scattering of the nanoparticles can be engineered by doping lanthanide ions, which is based on the enhanced polarizability of the nanoparticles when the excitation wavelength matches the ion resonance frequency of the lanthanide ions. More details can be found in article number 2203354 by Baolei Liu, Chaohao Chen, Xiaoxue Xu, Fan Wang, and co-workers.

PiSCAT: A Python Package for Interferometric Scattering Microscopy

PiSCAT: A Python Package for Interferometric Scattering Microscopy

TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data.

Single particle tracking (SPT) is one of the most widely used tools in optical microscopy to evaluate particle mobility in a variety of situations, including cellular and model membrane dynamics. Recent technological developments, such as Interferometric Scattering microscopy, have allowed recording of long, uninterrupted single particle trajectories at kilohertz framerates. The resulting data, where particles are continuously detected and do not displace much between observations, thereby do not require complex linking algorithms. Moreover, while these measurements offer more details into the short-term diffusion behaviour of the tracked particles, they are also subject to the influence of localisation uncertainties, which are often underestimated by conventional analysis pipelines. we thus developed a Python library, under the name of TRAIT2D (Tracking Analysis Toolbox - 2D version), in order to track particle diffusion at high sampling rates, and analyse the resulting trajectories with an innovative approach. The data analysis pipeline introduced is more localisation-uncertainty aware, and also selects the most appropriate diffusion model for the data provided on a statistical basis. A trajectory simulation platform also allows the user to handily generate trajectories and even synthetic time-lapses to test alternative tracking algorithms and data analysis approaches. A high degree of customisation for the analysis pipeline, for example with the introduction of different diffusion modes, is possible from the source code. Finally, the presence of graphical user interfaces lowers the access barrier for users with little to no programming experience.

TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data

Single particle tracking (SPT) is one of the most widely used tools in optical microscopy to evaluate particle mobility in a variety of situations, including cellular and model membrane dynamics. Recent technological developments, such as Interferometric Scattering microscopy, have allowed recording of long, uninterrupted single particle trajectories at kilohertz framerates. The resulting data, where particles are continuously detected and do not displace much between observations, thereby do not require complex linking algorithms. Moreover, while these measurements offer more details into the short-term diffusion behaviour of the tracked particles, they are also subject to the influence of localisation uncertainties, which are often underestimated by conventional analysis pipelines. we thus developed a Python library, under the name of TRAIT2D (Tracking Analysis Toolbox – 2D version), in order to track particle diffusion at high sampling rates, and analyse the resulting trajectories with an innovative approach. The data analysis pipeline introduced is more localisation-uncertainty aware, and also selects the most appropriate diffusion model for the data provided on a statistical basis. A trajectory simulation platform also allows the user to handily generate trajectories and even synthetic time-lapses to test alternative tracking algorithms and data analysis approaches. A high degree of customisation for the analysis pipeline, for example with the introduction of different diffusion modes, is possible from the source code. Finally, the presence of graphical user interfaces lowers the access barrier for users with little to no programming experience.

TRAIT2D: a Software for Quantitative Analysis of Single Particle Diffusion Data.

Single particle tracking (SPT) is one of the most widely used tools in optical microscopy to evaluate particle mobility in a variety of situations, including cellular and model membrane dynamics. Recent technological developments, such as Interferometric Scattering microscopy, have allowed recording of long, uninterrupted single particle trajectories at kilohertz framerates. The resulting data, where particles are continuously detected and do not displace much between observations, thereby do not require complex linking algorithms. Moreover, while these measurements offer more details into the short-term diffusion behaviour of the tracked particles, they are also subject to the influence of localisation uncertainties, which are often underestimated by conventional analysis pipelines. we thus developed a Python library, under the name of TRAIT2D (Tracking Analysis Toolbox - 2D version), in order to track particle diffusion at high sampling rates, and analyse the resulting trajectories with an innovative approach. The data analysis pipeline introduced is more localisation-uncertainty aware, and also selects the most appropriate diffusion model for the data provided on a statistical basis. A trajectory simulation platform also allows the user to handily generate trajectories and even synthetic time-lapses to test alternative tracking algorithms and data analysis approaches. A high degree of customisation for the analysis pipeline, for example with the introduction of different diffusion modes, is possible from the source code. Finally, the presence of graphical user interfaces lowers the access barrier for users with little to no programming experience.

High-Precision Protein-Tracking With Interferometric Scattering Microscopy.

The mobility of proteins and lipids within the cell, sculpted oftentimes by the organization of the membrane, reveals a great wealth of information on the function and interaction of these molecules as well as the membrane itself. Single particle tracking has proven to be a vital tool to study the mobility of individual molecules and unravel details of their behavior. Interferometric scattering (iSCAT) microscopy is an emerging technique well-suited for visualizing the diffusion of gold nanoparticle-labeled membrane proteins to a spatial and temporal resolution beyond the means of traditional fluorescent labels. We discuss the applicability of interferometric single particle tracking (iSPT) microscopy to investigate the minutia in the motion of a protein through measurements visualizing the mobility of the epidermal growth factor receptor in various biological scenarios on the live cell.

Emergence and Rearrangement of Dynamic Supramolecular Aggregates Visualized by Interferometric Scattering Microscopy.

Studying dynamic self-assembling systems in their native environment is essential for understanding the mechanisms of self-assembly and thereby exerting full control over these processes. Traditional ensemble-based analysis methods often struggle to reveal critical features of the self-assembly that occur at the single particle level. Here, we describe a label-free single-particle assay to visualize real-time self-assembly in aqueous solutions by interferometric scattering microscopy. We demonstrate how the assay can be applied to biphasic reactions yielding micellar or vesicular aggregates, detecting the onset of aggregate formation, quantifying the kinetics at the single particle level, and distinguishing sigmoidal and exponential growth of aggregate populations. Furthermore, we can follow the evolution in aggregate size in real time, visualizing the nucleation stages of the self-assembly processes and record phenomena such as incorporation of oily components into the micelle or vesicle lumen.

Myosin II Filament Dynamics in Actin Networks Revealed with Interferometric Scattering Microscopy

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood because of difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect and image individual membrane-attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network.

Interferometric Scattering Microscopy to Characterize Nanometric Objects and Subcellular Structures: Towards Fast 3D Imaging at Nanoscale

It is of great interest to visualize nano-scale objects in bioscience. Several ingenious methods such as super-resolution microscopic techniques have been developed to meet the need. These techniques, however, bear evident limitations in measurement time, time resolution, and requirement for fluorescent proteins or probes. In order to circumvent such problems, a new scattering-based method named iSCAT (interferometric scattering microscopy) has been recently introduced. Since this technique relies on scattering signal from a target particle and detects the interference of the signal with a constant reference, it is, in principle, not subject to the aforementioned limitations: theoretically unlimited signal-to-noise ratio to improve time resolution, practically unlimited measurement time, and lack of photophysical and photochemical artefacts.In this presentation, first, we show our recent application of this technique to image biological cells.Interestingly, the iSCAT technique enables us to visualize subcellular structures with remarkable spatial, temporal resolutions and contrast without any labeling.Second, to characterize the position and orientation of nanorods, we utilized scattering of polarized light from anisotropic scatterers, the strength of which is a function of relative orientation of the scatterers to the direction of polarization. From this scheme named psiSCAT (polarization selective iSCAT), we successfully demonstrate that one can capture the orientation of anisotropic nanorods without sacrificing the bright-field image of the entire view-field. Third, we can track the position of nanoparticles along the axial dimension and to image the sample at different depth with minimal image distortion by adopting the remote-focusing technique. We believe that the adoption of the remote-focusing approach significantly expands the strength and utility of iSCAT. This scattering-based microscopy technique would be an indispensable tool in visualizing the nanoscopic world and shed new light on phenomena occurring in the nanospace.

Dynactin p150 Promotes Processive Motility of DDB Complexes by Minimizing Diffusional Behavior of Dynein

Abstract Cytoplasmic dynein is activated by forming a complex with dynactin and the adaptor protein BicD2. We used Interferometric Scattering (iSCAT) microscopy to track dynein-dynactin-BicD2 (DDB) complexes in vitro and developed a regression-based algorithm to classify switching between processive, diffusive and stuck motility states. We find that DDB spends 65% of its time undergoing processive stepping, 4% undergoing 1D diffusion, and the remaining time transiently stuck to the microtubule. Although the p150 subunit was previously shown to enable dynactin diffusion along microtubules, blocking p150 enhanced the proportion of time DDB diffused and reduced the time DDB processively walked. Thus, DDB diffusive behavior most likely results from dynein switching into an inactive (diffusive) state, rather than p150 tethering the complex to the microtubule. DDB - kinesin-1 complexes, formed using a DNA adapter, moved slowly and persistently, and blocking p150 led to a 70 nm/s plus-end shift in the average velocity, in quantitative agreement with the increase in diffusivity seen in isolated DDB. The data suggest a DDB activation model in which engagement of dynactin p150 with the microtubule promotes dynein processivity, serves as an allosteric activator of dynein, and enhances processive minus-end motility during intracellular bidirectional transport. TOC Highlight Dynein-dynactin-BicD2 (DDB) is highly processive, but also shows transient pausing and diffusion, which we analyzed using iSCAT microscopy. Blocking dynactin p150 results in more diffusion of isolated DDB and a plus-end shift of kinesin-1 – DDB complexes. Thus, we conclude that p150 is an allosteric activator of dynein in the DDB complex.

Detection and Sizing of Submicron Particles in Biologics With Interferometric Scattering Microscopy

We demonstrate the application of interferometric scattering microscopy (IFS) for characterizing submicron particles in stir-stressed monoclonal antibody. IFS uses a layered silicon sensor and modified optical microscope to rapidly visualize and determine the particle size distribution (PSD) of submicron particles based on their scattering intensity, which is directly proportional to particle mass. Limits for particle size and optimal solution concentration were established for IFS characterization of submicron particles. We critically compare IFS data with dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) and find IFS is superior to NTA and DLS for determining the realistic shape of the number-based PSD, whereas NTA and DLS provide superior information about absolute particle size. Together, IFS, NTA, and DLS provide complementary information on submicron particles and enable quantitative characterization of the PSD of submicron aggregates. Finally, we explore quantifying particle size with IFS by developing a calibration curve for particle scattering intensity based on correlative scanning electron microscopy imaging. We found that only a subset of isotropic-shaped particles followed the expected proportionality between IFS intensity and particle mass. Overall, this study demonstrates IFS is a simple approach for detecting and quantifying submicron aggregate PSD in protein-based therapeutics.

Localization Error and Fitting Model Evaluation in Single Particle Tracking

Single Particle Tracking (SPT) is a powerful technique for uncovering molecular dynamics in lipid membrane biophysics, as well as in many other fields. Thus far, it has been successfully used to discover non-Brownian motion phenomena such as transient confined (also known as hop) diffusion of lipids and membrane proteins in the plasma membrane by accessing high framerates (103-104 fps) and claimed localization accuracies in the nanometre range. However, most of literature reporting high-speed SPT data relies on analysis protocols that are very susceptible to underestimations of the effects of localization errors; whose consequences are particularly pronounced at high framerates. Therefore, we propose a different approach to estimate such errors, and a statistically-driven procedure for determining the appropriate mode of motion fitting model. Using Interferometric Scattering (iSCAT) microscopy, we were able to collect high sampling frequency data of immobile and moving particles in different situations, to use for our analysis. First, we compare the effect of Signal-to-Noise Ratio on the localization precision of different immobile particles (gold nanoparticles, single proteins). Next, we show the effect of camera blur and localization error on the same particles diffusing on quasi-homogeneous Supported Lipid Bilayers (SLB), to develop an error correction method and a fitting goodness discrimination method of fitting SPT data. These last results are compared to Fluorescence Correlation Spectroscopy (FCS) measurements as a control for the values of diffusion rate. Finally, we acquired data of gold nanoparticle-tagged lipids diffusing on epithelial cell membranes, and apply the newly developed method to these data, to determine which model is the best statistical fit for cell membrane dynamics. In conclusion, the analysis workflow we propose, based on statistical methods rather than previous knowledge, could increase reliability and accuracy of future SPT measurements at high spatiotemporal resolution.

Revealing Compartmentalized Diffusion in Living Cells with Interferometric Scattering Microscopy

The spatiotemporal organization and dynamics of the plasma membrane and its constituents are central to cellular function. Fluorescence-based single-particle tracking has emerged as a powerful approach for studying the single molecule behavior of plasma-membrane-associated events because of its excellent background suppression, at the expense of imaging speed and observation time. Here, we show that interferometric scattering microscopy combined with 40 nm gold nanoparticle labeling can be used to follow the motion of membrane proteins in the plasma membrane of live cultured mammalian cell lines and hippocampal neurons with up to 3 nm precision and 25 μs temporal resolution. The achievable spatiotemporal precision enabled us to reveal signatures of compartmentalization in neurons likely caused by the actin cytoskeleton.

Variation in Stride Length of Myosin-5A Revealed by Interferometric Scattering Microscopy (iSCAT)

Variation in Stride Length of Myosin-5A Revealed by Interferometric Scattering Microscopy (iSCAT)

Label-Free Single-Molecule Imaging with Numerical-Aperture-Shaped Interferometric Scattering Microscopy.

Our ability to optically interrogate nanoscopic objects is controlled by the difference between their extinction cross sections and the diffraction-limited area to which light can be confined in the far field. We show that a partially transmissive spatial mask placed near the back focal plane of a high numerical aperture microscope objective enhances the extinction contrast of a scatterer near an interface by approximately T–1/2, where T is the transmissivity of the mask. Numerical-aperture-based differentiation of background from scattered light represents a general approach to increasing extinction contrast and enables routine label-free imaging down to the single-molecule level.

High-Speed Nanometric Tracking of Myosin 5 with Interferometric Scattering Microscopy

Myosin 5 is a molecular motor that moves along actin filaments by taking 74 nm steps. Although its enzymatic cycle is well understood, there is inconclusive and conflicting information on the motion that describes individual steps. Studies aimed at revealing the stepping mechanism have either reported periods of increased flexibility or proposed partitioning of the step into one or multiple sub-events. All resulting models involve a forward aiming power stroke followed by a Brownian search of the unbound head. We used interferometric scattering microscopy (iSCAT) to track the head of myosin 5 with simultaneous nanometer spatial and millisecond temporal precision. We observed a single, spatially constrained transient state of the detached head. Within individual traces, the position of the transient always occurred on the same side of actin filament but the sidedness could change when myosin molecule switched tracks. Simultaneous tracking of both heads revealed that myosin moves in a “spinning” hand-over-hand fashion, where both heads follow the same path and where torsional strain plays an important role. We also show that, in contradiction to the Brownian search hypothesis, the detached head reaches the desired binding site in a highly controlled manner. Improving the spatial precision to the sub-nm regime revealed a structural transition in the motor domain associated with the power stroke in the leading head, which may correspond to strain release. Taken together, our results demonstrate how the motor structure tightly controls its movement.

Label-Free Imaging of Microtubules with Subnanometer Precision using Interferometric Scattering Microscopy

Label-Free Imaging of Microtubules with Subnanometer Precision using Interferometric Scattering Microscopy

Label-free Imaging of Microtubules with Sub-nm Precision Using Interferometric Scattering Microscopy

Current in vitro optical studies of microtubule dynamics tend to rely on fluorescent labeling of tubulin, with tracking accuracy thereby limited by the quantum yield of fluorophores and by photobleaching. Here, we demonstrate label-free tracking of microtubules with nanometer precision at kilohertz frame rates using interferometric scattering microscopy (iSCAT). With microtubules tethered to a glass substrate using low-density kinesin, we readily detect sequential 8 nm steps in the microtubule center of mass, characteristic of a single kinesin molecule moving a microtubule. iSCAT also permits dynamic changes in filament length to be measured with <5 nm precision. Using the arbitrarily long observation time enabled by label-free iSCAT imaging, we demonstrate continuous monitoring of microtubule disassembly over a 30 min period. The ability of iSCAT to track microtubules with nm precision together with its potential for label-free single protein detection and simultaneous single molecule fluorescence imaging represent a unique platform for novel approaches to studying microtubule dynamics.