Development and applications of single particle orientation and rotational tracking in dynamic systems

Optical microscopy has successfully been used to visualize the dynamics of living systems for decades. Numerous microscopic techniques, including single particle tracking (SPT), have been developed to measure dynamic processes in living cells with minimum disruption to the cellular functions. SPT is capable of accessing individual behaviors of imaging probe (single molecule or nanoparticle) with high spatial and temporal resolution. It has been utilized for investigating dynamic events of single molecule and nanoparticle in many different biological systems. Single particle orientation and rotational tracking (SPORT) studies not only the spatial movements as in conventional SPT but also the orientation and rotational behavior of imaging probes in order to reveal the molecular mechanisms involved in fundamental motions. A variety of experimental techniques have been reported for determining the orientation and rotational motions of optical imaging probes. Differential interference contrast (DIC) microscopy, along with the use of anisotropic plasmonic nanoparticles as optical probes, offers unique ability in SPORT study. Synthesis of novel imaging probes, innovations in optical implementation, advance in data analysis could all contribute to the development and expanding the applications of SPORT techniques in dynamic studies. Multishell Au/Ag/SiO2 core-shell hybrid nanorods with tunable optical properties were synthesized and used as new SPORT probes in DIC microscopy. These nanorods provided enhanced detection sensitivity, improved stability and additional surface modification possibility. The addition of a wedge prism and the implementation of auto-focusing algorithm formed Parallax-DIC microscopy in the 5D-SPT method. It enables the simultaneous 3D spatial tracking and orientation determination in visualization of intracellular transport of cargos in live cells. Autocorrelation function analysis and binning function in imageJ was utilized for DIC data processing. Experimental parameters, such as trajectory length of the SPT tracking and frame rate (exposure time) were investigated for the rotation of surface modified gold nanorods on synthetic lipid bilayers with assistance of computer simulations. However, much effort is still required in exploring new imaging probes, optical microscopic implementations, and data analysis methods to further extend the potential of DIC-based SPORT techniques in dynamic studies

Coordinates in low-dimensional cell shape-space discriminate migration dynamics from single static cell images

Cell shape has long been used to discern cell phenotypes and states, but the underlying premise has not been quantitatively tested. Here, we show that a single cell image can be used to discriminate its migration behavior by analyzing a large number of cell migration data in vitro. We analyzed a large number of two-dimensional cell migration images over time and found that the cell shape variation space has only six dimensions, and migration behavior can be determined by the coordinates of a single cell image in this 6-dimensional shape-space. We further show that this is possible because persistent cell migration is characterized by spatial-temporally coordinated protrusion and contraction, and a distribution signature in the shape-space. Our findings provide a quantitative underpinning for using cell morphology to differentiate cell dynamical behavior.Comment: 29 pages, 9 figure

Multishell Au/Ag/SiO2 Nanorods with Tunable Optical Properties as Single Particle Orientation and Rotational Tracking Probes

Three-layer core-shell plasmonic nanorods (Au/Ag/SiO2-NRs), consisting of a gold nanorod core, a thin silver shell, and a thin silica layer, were synthesized and used as optical imaging probes under a differential interference contrast microscope for single particle orientation and rotational tracking. The localized surface plasmon resonance modes were enhanced upon the addition of the silver shell, and the anisotropic optical properties of gold nanorods were maintained. The silica coating enables surface functionalization with silane coupling agents and provides enhanced stability and biocompatibility. Taking advantage of the longitudinal LSPR enhancement, the orientation and rotational information of the hybrid nanorods on synthetic lipid bilayers and on live cell membranes were obtained with millisecond temporal resolution using a scientific complementary metal-oxide-semiconductor camera. The results demonstrate that the as-synthesized hybrid nanorods are promising imaging probes with improved sensitivity and good biocompatibility for single plasmonic particle tracking experiments in biological systems

Development and applications of single particle orientation and rotational tracking in dynamic systems

Optical microscopy has successfully been used to visualize the dynamics of living systems for decades. Numerous microscopic techniques, including single particle tracking (SPT), have been developed to measure dynamic processes in living cells with minimum disruption to the cellular functions. SPT is capable of accessing individual behaviors of imaging probe (single molecule or nanoparticle) with high spatial and temporal resolution. It has been utilized for investigating dynamic events of single molecule and nanoparticle in many different biological systems. Single particle orientation and rotational tracking (SPORT) studies not only the spatial movements as in conventional SPT but also the orientation and rotational behavior of imaging probes in order to reveal the molecular mechanisms involved in fundamental motions. A variety of experimental techniques have been reported for determining the orientation and rotational motions of optical imaging probes. Differential interference contrast (DIC) microscopy, along with the use of anisotropic plasmonic nanoparticles as optical probes, offers unique ability in SPORT study. Synthesis of novel imaging probes, innovations in optical implementation, advance in data analysis could all contribute to the development and expanding the applications of SPORT techniques in dynamic studies. Multishell Au/Ag/SiO2 core-shell hybrid nanorods with tunable optical properties were synthesized and used as new SPORT probes in DIC microscopy. These nanorods provided enhanced detection sensitivity, improved stability and additional surface modification possibility. The addition of a wedge prism and the implementation of auto-focusing algorithm formed Parallax-DIC microscopy in the 5D-SPT method. It enables the simultaneous 3D spatial tracking and orientation determination in visualization of intracellular transport of cargos in live cells. Autocorrelation function analysis and binning function in imageJ was utilized for DIC data processing. Experimental parameters, such as trajectory length of the SPT tracking and frame rate (exposure time) were investigated for the rotation of surface modified gold nanorods on synthetic lipid bilayers with assistance of computer simulations. However, much effort is still required in exploring new imaging probes, optical microscopic implementations, and data analysis methods to further extend the potential of DIC-based SPORT techniques in dynamic studies.</p

Multishell Au/Ag/SiO2 Nanorods with Tunable Optical Properties as Single Particle Orientation and Rotational Tracking Probes

Three-layer core-shell plasmonic nanorods (Au/Ag/SiO2-NRs), consisting of a gold nanorod core, a thin silver shell, and a thin silica layer, were synthesized and used as optical imaging probes under a differential interference contrast microscope for single particle orientation and rotational tracking. The localized surface plasmon resonance modes were enhanced upon the addition of the silver shell, and the anisotropic optical properties of gold nanorods were maintained. The silica coating enables surface functionalization with silane coupling agents and provides enhanced stability and biocompatibility. Taking advantage of the longitudinal LSPR enhancement, the orientation and rotational information of the hybrid nanorods on synthetic lipid bilayers and on live cell membranes were obtained with millisecond temporal resolution using a scientific complementary metal-oxide-semiconductor camera. The results demonstrate that the as-synthesized hybrid nanorods are promising imaging probes with improved sensitivity and good biocompatibility for single plasmonic particle tracking experiments in biological systems.Reprinted (adapted) with permission from Analytical Chemistry 87 (2015): 4096, doi: 10.1021/acs.analchem.5b00604. Copyright 2015 American Chemical Society.</p

Smartphone-Based Portable Bio-Chemical Sensors: Exploring Recent Advancements

Traditionally, analytical chemistry and diagnosis relied on wet laboratories and skilled professionals utilizing sophisticated instruments for sample handling and analysis. However, with the development of novel materials and sensing techniques, there has been a significant shift towards the use of standalone sensors, allowing tests to be conducted on-site or even in real time, leading to cost- and time-efficiency. With their widespread adoption globally, smartphones have emerged as an ideal platform for such sensors, boasting extensive sensor capabilities, advanced processing power, and communication functionalities. Smartphone-based assays make use of optical and electrochemical sensors, utilizing built-in cameras, ambient light sensors, and other features for optical sensing, while the micro-USB port, Bluetooth, and wireless connection facilitate data transmission and analog voltage application for electrochemical sensing. Previous overview papers have explored smartphone-based sensing in specific domains; this review provides a comprehensive examination of recent advancements in smartphone-based sensors, encompassing both optical and electrochemical sensing methods. The review provides the fundamental principles of these sensors and their implementation using smartphones, showcases recent applications, and presents innovative designs that take advantage of the inherent functionalities and sensor capabilities of smartphones. The review concludes by offering an outlook on the prospects of smartphone-based sensing and includes a reflective section emphasizing the potential impact of sensors in chemical and biological analyses. This comprehensive resource aims to provide information to researchers and practitioners interested in using smartphones for cutting-edge analytical methodologies

Characteristic rotational behaviors of rod-shaped cargo revealed by automated five-dimensional single particle tracking.

We report an automated single particle tracking technique for tracking the x, y, z coordinates, azimuthal and elevation angles of anisotropic plasmonic gold nanorod probes in live cells. These five spatial coordinates are collectively referred to as 5D. This method overcomes a long-standing challenge in distinguishing rotational motions from translational motions in the z-axis in differential interference contrast microscopy to result in full disclosure of nanoscale motions with high accuracy. Transferrin-coated endocytic gold nanorod cargoes initially undergo active rotational diffusion and display characteristic rotational motions on the membrane. Then as the cargoes being enclosed in clathrin-coated pits, they slow down the active rotation and experience a quiet period before they restore active rotational diffusion after fission and eventually being transported away from the original entry spots. Finally, the 3D trajectories and the accompanying rotational motions of the cargoes are resolved accurately to render the intracellular transport process in live cells.Distinguishing rotational motions from translational motions in the z-axis has been a long-standing challenge. Here the authors develop a five-dimensional single particle tracking method to detect rotational behaviors of nanocargos during clathrin-mediated endocytosis and intracellular transport

Characteristic rotational behaviors of rod-shaped cargo revealed by automated five-dimensional single particle tracking

Distinguishing rotational motions from translational motions in the z-axis has been a long-standing challenge. Here the authors develop a five-dimensional single particle tracking method to detect rotational behaviors of nanocargos during clathrin-mediated endocytosis and intracellular transport

Gold Nanorod Photothermal Therapy Alters Cell Junctions and Actin Network in Inhibiting Cancer Cell Collective Migration

Most cancer-related deaths come from metastasis. It was recently discovered that nanoparticles could inhibit cancer cell migration. Whereas most researchers focus on single-cell migration, the effect of nanoparticle treatment on collective cell migration has not been explored. Collective migration occurs commonly in many types of cancer metastasis, where a group of cancer cells move together, which requires the contractility of the cytoskeleton filaments and the connection of neighboring cells by the cell junction proteins. Here, we demonstrate that gold nanorods (AuNRs) and the introduction of near-infrared light could inhibit the cancer cell collective migration by altering the actin filaments and cell junctions with significantly triggered phosphorylation changes of essential proteins, using mass spectrometry-based phosphoproteomics. Further observation using super-resolution stochastic optical reconstruction microscopy (STORM) showed the actin cytoskeleton filament bundles were disturbed, which is difficult to differentiate under a normal fluorescence microscope. The decreased expression level of N-cadherin junctions and morphological changes of tight junction protein zonula occludens 2 were also observed. All of these results indicate possible functions of the AuNR treatments in regulating and remodeling the actin filaments and cell junction proteins, which contribute to decreasing cancer cell collective migration