Molecular machines open cell membranes.

Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

Resolving cargo-motor-track interactions with bifocal parallax single-particle tracking.

Resolving coordinated biomolecular interactions in living cellular environments is vital for understanding the mechanisms of molecular nanomachines. The conventional approach relies on localizing and tracking target biomolecules and/or subcellular organelles labeled with imaging probes. However, it is challenging to gain information on rotational dynamics, which can be more indicative of the work done by molecular motors and their dynamic binding status. Herein, a bifocal parallax single-particle tracking method using half-plane point spread functions has been developed to resolve the full-range azimuth angle (0-360°), polar angle, and three-dimensional (3D) displacement in real time under complex living cell conditions. Using this method, quantitative rotational and translational motion of the cargo in a 3D cell cytoskeleton was obtained. Not only were well-known active intracellular transport and free diffusion observed, but new interactions (tight attachment and tethered rotation) were also discovered for better interpretation of the dynamics of cargo-motor-track interactions at various types of microtubule intersections.

Developing Noise-Resistant Three-Dimensional Single Particle Tracking Using Deep Neural Networks.

Three-dimensional single particle tracking (3D SPT) is a powerful tool in various chemical and biological studies. In 3D SPT, z sensitive point spread functions (PSFs) are frequently used to generate different patterns, from which the axial position of the probe can be recovered in addition to its xy coordinates. Conventional linear classifier-based methods, for example, the correlation coefficient method, perform poorly when the signal-to-noise ratio (S/N) drops. In this work, we test deep neural networks (DNNs) in recognizing and differentiating very similar image patterns incurred in 3D SPT. The training of the deep neural networks is optimized, and a procedure is established for 3D localization. We show that for high S/N images, both DNNs and conventional correlation coefficient-based method perform well. However, when the S/N drops close to 1, conventional methods completely fail while DNNs show strong resistance to both artificial and experimental noises. This noise resistance allows us to achieve a camera integration time of 50 µs for 200 nm fluorescent particles without losing accuracy significantly. This study sheds new light on developing robust image data analysis methods and on improving the time resolution of 3D SPT.

Three-Dimensional Heterogeneous Structure Formation on a Supported Lipid Bilayer Disclosed by Single-Particle Tracking.

Three-dimensional (3D) single-particle tracking was employed to study the lipid membrane morphology change at different pHs on glass supported lipid bilayers (SLBs) [1,2-dioleoyl- sn-glycero-3-phosphoethanolamine/1,2-dioleoyl- sn-glycero-3-phospho-l-serine (sodium salt)/1,2-dioleoyl- sn-glycero-3-phosphocholine = 5:3:2]. Fluorescently tagged, carboxylated polystyrene nanoparticles (of 100 nm) were used as the probes. At neutral pHs, the particles' diffusion was close to two-dimensional Brownian motion, indicating a mainly planar structure of the SLBs. When the environmental pH was tuned to be basic at 10.0, transiently confined diffusions within small areas were frequently observed. These confinements had a lateral dimension of 100-200 nm. Most interestingly, they showed 3D bulged structures protruding from the planar lipid bilayer. The particles were trapped by these 3D structures for a short period of time (∼0.75 s), with an estimated escape activation energy of ∼4.2 kB T. Nonuniform distribution of pH-sensitive lipids in the membrane was proposed to explain the formation of these 3D heterogeneous structures. This work suggests that the geometry of the 3D lipid structures can play a role in tuning the particle-lipid surface interactions. It sheds new light on the origin of lateral heterogeneity on the lipid membrane.

Microscopic Movement of Slow-Diffusing Nanoparticles in Cylindrical Nanopores Studied with Three-Dimensional Tracking.

To study slow mass transport in confined environments, we developed a three-dimensional (3D) single-particle localization technique to track their microscopic movements in cylindrical nanopores. Under two model conditions, particles are retained much longer inside the pores: (1) increased solvent viscosity, which slows down the particle throughout the whole pore, and (2) increased pore wall affinity, which slows down the particle only at the wall. In viscous solvents, the particle steps decrease proportionally to the increment of the viscosity, leading to macroscopically slow diffusion. As a contrast, the particles in sticky pores are microscopically active by showing limited reduction of step sizes. A restricted diffusion mode, possibly caused by the heterogeneous environment in sticky pores, is the main reason for macroscopically slow diffusion. This study shows that it is possible to differentiate slow diffusion in confined environments caused by different mechanisms.

Investigating axial diffusion in cylindrical pores using confocal single-particle fluorescence correlation spectroscopy.

We explored the feasibility of using confocal fluorescence correlation spectroscopy to study small nanoparticle diffusion in hundred-nanometer-sized cylindrical pores. By modeling single particle diffusion in tube-like confined three-dimensional space aligned parallel to the confocal optical axis, we showed that two diffusion dynamics can be observed in both original intensity traces and the autocorrelation functions (ACFs): the confined two-dimensional lateral diffusion and the unconfined one-dimensional (1D) axial diffusion. The separation of the axial and confined lateral diffusion dynamics provides an opportunity to study diffusions in different dimensions separately. We further experimentally studied 45 nm carboxylated polystyrene particles diffusing in 300 nm alumina pores. The experimental data showed consistency with the simulation. To extract the accurate axial diffusion coefficient, we found that a 1D diffusion model with a Lorentzian axial collection profile needs to be used to analyze the experimental ACFs. The diffusion of the 45 nm nanoparticles in polyethyleneglycol-passivated 300 nm pores slowed down by a factor of ∼2, which can be satisfactorily explained by hydrodynamic frictions.

Nanosecond Time-Resolution Study of Gold Nanorod Rotation at the Liquid-Solid Interface.

Early studies showed that the adsorption of nanorods may start from a special "anchored" state, in which the nanorods lose translational motion but retain rotational freedom. Insight into how the anchored nanorods rotate should provide additional dimensions for understanding particle-surface interactions. Based on conventional time-resolution studies, gold nanorods are thought to continuously rotate following initial interactions with negatively charged glass surfaces. However, this nanosecond time-resolution study reveals that the apparent continuous rotation actually consists of numerous fast, intermittent rotations or transitions between a small number of weakly immobilized states, with the particle resting in the immobilized states most of the time. The actual rotation from one immobilized state to the other happens on a 1 ms timescale, that is, approximately 50 times slower than in the bulk solution.

Unimolecular Submersible Nanomachines. Synthesis, Actuation, and Monitoring.

Unimolecular submersible nanomachines (USNs) bearing light-driven motors and fluorophores are synthesized. NMR experiments demonstrate that the rotation of the motor is not quenched by the fluorophore and that the motor behaves in the same manner as the corresponding motor without attached fluorophores. No photo or thermal decomposition is observed. Through careful design of control molecules with no motor and with a slow motor, we found using single molecule fluorescence correlation spectroscopy that only the molecules with fast rotating speed (MHz range) show an enhancement in diffusion by 26% when the motor is fully activated by UV light. This suggests that the USN molecules give ∼9 nm steps upon each motor actuation. A non-unidirectional rotating motor also results in a smaller, 10%, increase in diffusion. This study gives new insight into the light actuation of motorized molecules in solution.

A temperature microsensor for measuring laser-induced heating in gold nanorods.

Measuring temperature is an extensively explored field of analysis, but measuring a temperature change in a nanoparticle is a new challenge. Here, a microsensor is configured to measure temperature changes in gold nanorods in solution upon laser irradiation. The device consists of a silicon wafer coated with silicon nitride in which a microfabricated resistance temperature detector was embedded and attached to a digital multimeter. A polydimethylsiloxane mold served as a microcontainer for the sample attached on top of the silicon membrane. This enables laser irradiation of the gold nanorods and subsequent measurement of temperature changes. The results showed a temperature increase of 8 to 10 °C and good correlation with theoretical calculations and bulk sample direct temperature measurements. These results demonstrate the suitability of this simple temperature microsensor for determining laser-induced heating profiles of metallic nanomaterials; such measurements will be essential for optimizing therapeutic and catalytic applications.

Continuous-Wave Stimulated Emission Depletion Microscope for Imaging Actin Cytoskeleton in Fixed and Live Cells.

Stimulated emission depletion (STED) microscopy provides a new opportunity to study fine sub-cellular structures and highly dynamic cellular processes, which are challenging to observe using conventional optical microscopy. Using actin as an example, we explored the feasibility of using a continuous wave (CW)-STED microscope to study the fine structure and dynamics in fixed and live cells. Actin plays an important role in cellular processes, whose functioning involves dynamic formation and reorganization of fine structures of actin filaments. Frequently used confocal fluorescence and STED microscopy dyes were employed to image fixed PC-12 cells (dyed with phalloidin- fluorescein isothiocyante) and live rat chondrosarcoma cells (RCS) transfected with actin-green fluorescent protein (GFP). Compared to conventional confocal fluorescence microscopy, CW-STED microscopy shows improved spatial resolution in both fixed and live cells. We were able to monitor cell morphology changes continuously; however, the number of repetitive analyses were limited primarily by the dyes used in these experiments and could be improved with the use of dyes less susceptible to photobleaching. In conclusion, CW-STED may disclose new information for biological systems with a proper characteristic length scale. The challenges of using CW-STED microscopy to study cell structures are discussed.

Up-conversion luminescence of gold nanospheres when excited at nonsurface plasmon resonance wavelength by a continuous wave laser.

We show that, when gold nanospheres are excited at the red side of the surface plasmon resonance (SPR) wavelength at 592 nm by a continuous wave (CW) laser, they give substantial up-converted luminescence in the SPR wavelength range. The luminescence intensity scales as a second-order function of the excitation power, with a quantum yield ~1/50 of down-conversion luminescence when illuminated at a power of 30 MW/cm(2). The luminescence spectrum is completely different than the SPR profile, indicating a new emission mechanism possibly involving interband transitions coupled with phonons or localized vibration of neighboring gold atoms. Such luminescence is also observed to be substantial for short gold nanorods with an aspect ratio of ~2 but weak for bulk gold. This study provides new insight to the understanding of gold nanoparticle luminescence and opens a new detection scheme for gold nanoparticle-based biological imaging.

Revealing rotational modes of functionalized gold nanorods on live cell membranes.

A full understanding of cell mechanics requires knowledge of both translational and rotational dynamics. The single particle orientation and rotational tracking (SPORT) technique is combined here with correlation analysis to identify the fundamental rotational modes: in-plane rotation and out-of-plane tilting, as well as other more complex rotational patterns, from the vast image data captured at a temporal resolution of 5 ms for single gold nanorod probes in live cell imaging experiments. The unique capabilities of visualizing and understanding rotational motions of functional nanoparticles on live cell membranes allow correlation of the rotational and translational dynamics in unprecedented detail and provide new insights into complex membrane processes. Particles with functionalized surfaces, which interact with the membrane in fundamentally different ways, can exhibit distinct rotational modes and are, for the first time, directly visualized, and these show the early events for membrane approach and attachment.

Dual-modality single particle orientation and rotational tracking of intracellular transport of nanocargos.

The single particle orientation and rotational tracking (SPORT) technique was introduced recently to follow the rotational motion of plasmonic gold nanorod under a differential interference contrast (DIC) microscope. In biological studies, however, cellular activities usually involve a multiplicity of molecules; thus, tracking the motion of a single molecule/object is insufficient. Fluorescence-based techniques have long been used to follow the spatial and temporal distributions of biomolecules of interest thanks to the availability of multiplexing fluorescent probes. To know the type and number of molecules and the timing of their involvement in a biological process under investigation by SPORT, we constructed a dual-modality DIC/fluorescence microscope to simultaneously image fluorescently tagged biomolecules and plasmonic nanoprobes in living cells. With the dual-modality SPORT technique, the microtubule-based intracellular transport can be unambiguously identified while the dynamic orientation of nanometer-sized cargos can be monitored at video rate. Furthermore, the active transport on the microtubule can be easily separated from the diffusion before the nanocargo docks on the microtubule or after it undocks from the microtubule. The potential of dual-modality SPORT is demonstrated for shedding new light on unresolved questions in intracellular transport.

Influence of polarization setting on gold nanorod signal at nonplasmonic wavelengths under differential interference contrast microscopy.

Researchers rely on a variety of microscopic techniques for observing and tracking anisotropic nanoparticles in real time experiments. This technical note focuses on the optical behavior exhibited by gold nanorods at nonplasmonic wavelengths under differential interference contrast microscopy (DIC). Intense diffraction patterns appear at nonplasmonic wavelengths, and the behavior of these patterns can be altered by adjusting the surrounding medium or the polarizer setting. Such patterns are absent when linear and crossed polarizations are utilized. Making polarization adjustments is important in DIC microscopy, because it affects bias retardation and image contrast. The nonplasmonic diffraction bands that were observed could potentially be exploited for rotational tracking, but more importantly, researchers should exhibit care in selecting a nanorod sample and the polarization setting when working with DIC microscopy.

Three-dimensional super-localization and tracking of single gold nanoparticles in cells.

We introduce a precise three-dimensional (3D) localization method of spherical gold nanoparticle probes using model-based correlation coefficient mapping. To accomplish this, a stack of sample images at different z-positions are acquired, and a 3D intensity profile of the probe serving as the model is used to map out the positions of nanoparticles in the sample. By using this model-based correlation imaging method, precise localization can be achieved in imaging techniques with complicated point spread functions (PSF) such as differential interference contrast (DIC) microscopy. We demonstrated the localization precision of 4-7 nm laterally and 16 nm axially for 40-nm gold nanospheres at an imaging rate of 10 frames per second. The 3D superlocalization method was applied to tracking gold nanospheres during live endocytosis events.

Determining the full three-dimensional orientation of single anisotropic nanoparticles by differential interference contrast microscopy.

Keeping track: By combining differential interference contrast (DIC) image pattern recognition with DIC polarization anisotropy, the exact full three-dimensional angular information of individual tilted gold nanorods positioned in the focal plane of the objective lens can be readily determined. The angular rotational modes and kinetics of individual in-focus gold nanorods can thus be resolved dynamically.

Single particle orientation and rotation tracking discloses distinctive rotational dynamics of drug delivery vectors on live cell membranes.

Engineered nanoparticles have emerged as potentially revolutionary drug and gene delivery vectors. Using rod-shaped gold nanoparticles as a model, we studied for the first time the rotational dynamics of nanoparticle vectors on live cell membranes and its impact on the fate of these nanoparticle vectors. The rotational motions of gold nanorods with various surface modifiers were tracked continuously at 200 frames/s under a differential interference contrast microscope. We found that the rotational behaviors of gold nanorod vectors are strongly related to their surface charges. Specific surface functional groups and the availability of receptors on cell membranes also contribute to the rotational dynamics. The study of rotational brownian motion of nanoparticles on cell membranes will lead to a better understanding of the mechanisms of drug delivery and provide guidance in designing surface modification strategies for drug delivery vectors under various circumstances.

Superlocalization of single molecules and nanoparticles in high-fidelity optical imaging microfluidic devices.

Superlocalization of single molecules and nanoparticles with a precision of subnanometer to a few tens of nanometers is crucial for elucidating nanoscale structures and movements in biological and chemical systems. A novel design of ultraflat and ultrathin glass/polydimethylsiloxane (PDMS) hybrid microdevices is introduced to provide almost uncompromised optical imaging quality for on-chip superlocalization and super-resolution imaging of single molecules and nanoparticles under a variety of microscopy modes. The performance of the high-fidelity (Hi-Fi) optical imaging microfluidic device was validated by precisely mapping micronecklaces made of fluorescent microtubules and 40 nm gold nanoparticles and by demonstrating the activation and excitation cycles of single Alexa Fluor 647 dyes for direct stochastic optical reconstruction microscopy in PDMS-based microchannels for the first time. Furthermore, the microdevice's feasibility for multimodality microscopy imaging was demonstrated by a vertical scan of live cells in epi-fluorescence and differential interference contrast (DIC) microscopy modes simultaneously.

Detection of the potential pancreatic cancer marker MUC4 in serum using surface-enhanced Raman scattering.

Pancreatic cancer (PC) is one of the most lethal malignancies. It has a 5-year survival rate of only 6%, owing in part to the lack of a reliable tumor marker for early diagnosis. Recent research has shown that the mucin protein MUC4 is aberrantly expressed in pancreatic adenocarcinoma cell lines and tissues but is undetectable in normal pancreas and chronic pancreatitis. Thus, the level of MUC4 in patient sera has the potential to function as a diagnostic and prognostic marker for PC. However, the measurement of MUC4 in sera using conventional test platforms (e.g., enzyme linked immunosorbent assay (ELISA) and radioimmunoassay (RIA)) has been unsuccessful. This has prevented the assessment of the utility of this protein as a possible PC marker in sera. In addressing this obstacle, the work herein examines the potential to create a simple diagnostic test for MUC4 through the development of a surface-enhanced Raman scattering (SERS)-based immunoassay, which was then used to demonstrate the first ever detection of MUC4 in cancer patient serum samples. Importantly, these measurements showed that sera from patients with PC produced a significantly higher SERS response for MUC4 compared to sera from healthy individuals and from patients with benign diseases. These results indicate that a SERS-based immunoassay can monitor MUC4 levels in patient sera, representing a much needed first step toward assessing the potential of this protein to serve as a serum marker for the early stage diagnosis of PC. This paper details these and other findings (i.e., the detection of the mucin protein CA19-9), which demonstrate that our SERS assay outperforms conventional assays (i.e., RIA and ELISA) with respect to limits of detection, readout time, and required sample volume.

Dynamin-dependent vesicle twist at the final stage of clathrin-mediated endocytosis.

Dynamin has an important role in clathrin-mediated endocytosis by cutting the neck of nascent vesicles from the cell membrane. Here, using gold nanorods as cargos to image dynamin action during live clathrin-mediated endocytosis, we show that, near the peak of dynamin accumulation, the cargo-containing vesicles always exhibit abrupt, right-handed rotations that finish in a short time (~0.28 s). The large and quick twist, herein named the super twist, is the result of the coordinated dynamin helix action upon GTP hydrolysis. After the super twist, the rotational freedom of the vesicle increases substantially, accompanied by simultaneous or delayed translational movement, indicating that it detaches from the cell membrane. These observations suggest that dynamin-mediated scission involves a large torque generated by the coordinated actions of multiple dynamins in the helix, which is the main driving force for vesicle scission.