Sub-millisecond Temporal Resolution Research Articles

3D Tracking of Cellular Nanoscale Dynamics with Sub-Millisecond Temporal Resolution

The study of highly dynamic cellular processes is leveraged by microscopic techniques with high temporal resolution. Especially for diffusive processes, where particle displacements can exceed 100 nm/ms, sub-millisecond temporal resolution is often required in addition to precise three-dimensional localization.We have developed a novel particle tracking microscope capable of observing fast 3D trajectories of fluorescently tagged intracellular objects with 300 μs temporal resolution [1]. Combining scanning-free biplane detection [2] with optimized beam steering, we were able to demonstrate 3D tracking of single fluorescent particles at speeds of up to 150 nm/ms over several seconds and large volumes. Maximum detection efficiency and minimal laser irradiation are guaranteed by focused excitation of only the particle of interest and by exploiting the high quantum efficiency of an EM-CCD camera. Limiting detection to a small subarea of the CCD chip improves speed and reduces background fluorescence. Combined with the absence of sample movement, these features ensure high live-sample compatibility.Here, we present recent improvements of the setup using fast adaptive optics for axial scanning. This enhances response times and eliminates mechanical coupling of moving components to the sample. We also show data from recent live-cell experiments. We have tracked actin-driven motion of GFP-labeled HIV-like particles on the cytoplasmic membrane. This “surfing” is involved in retroviral cell-to-cell spread [3] and precedes endocytosis. Fast 3D particle tracking has the potential to contribute to the understanding of the underlying mechanism.[1] M. F. Juette and J. Bewersdorf, Nano Lett., 10(11), 4657-4663 (2010).[2] M. F. Juette et al., Nat. Methods, 5(6), 527-529 (2008).[3] W. Mothes et al., J. Virol., 84(17), 8360-8368 (2010).

Ultra-fast in vivo x-ray microtomography of blowfly wingbeat

Ultra-fast in vivo x-ray microtomography of blowfly wingbeat

Single-Molecule Transport Across an Individual Biomimetic Nuclear Pore Complex

Nuclear pore complexes regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells. Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon. In this talk, I will show selective transport of proteins across individual biomimetic nuclear pore complexes at the single-molecule level. Each biomimetic complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanopore. Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (ImpB) proceed with a dwell time of ∼2.5 ms for both Nup98- and Nup153-coated pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For pores up to ∼25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger pores. Our biomimetic nuclear pore complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro. ∗ Work carried out by my graduate student Stefan W. Kowalczyk at Delft and in collaboration with Larisa Kapinos and Roderick Y. H. Lim at the University of Basel.

Dynamin-Related Protein-1 Controls Fusion Pore Dynamics During Platelet Granule Secretion and Thrombus Formation In Vivo

Abstract 361Platelet granule secretion serves a central role in hemostasis and thrombosis. During platelet secretion, fusion of granule membranes with those of the plasma membrane results in the release of granule contents. Recently electrochemical techniques using single-cell amperometry have shown that platelet membrane fusion results in the formation of a fusion pore. The fusion pore subsequently expands to enable the complete extrusion of granule contents. However, the molecular mechanisms that control platelet fusion pore formation and expansion are not known. To discover novel components of the platelet secretory machinery, we tested >300,000 compounds in a forward chemical genetic screen designed to identify inhibitors of dense granule secretion. A compound, ML160, was found that blocked dense granule release with an IC50 of approximately 0.5 μM. ML160 was also identified in an unrelated high throughput screen designed to detect inhibitors of dynamin-related protein-1 (Drp-1). Although best known as mediators of membrane fission, dynamins also contribute to granule exocytosis by controlling fusion pore expansion. Immunoblot analysis of platelet pellets and supernatants confirmed the presence of Drp-1 in platelets and demonstrated nearly equal distribution between platelet membranes and cytosol. mDivi-1, a well-characterized small molecule inhibitor of Drp-1 that acts outside of the GTP binding site, blocked PAR1-mediated platelet dense granule and α-granule release with an IC50 of approximately 20 μM. mDivi-1 also inhibited granule release induced by the thromboxane receptor agonist U46619, PMA, or Ca2+ ionophore, indicating that Drp-1 acts distally in the secretory pathway. To assess whether Drp-1 functions in platelet fusion pore dynamics, we tested the effect of mDivi-1 on the release of dense granules from rabbit platelets using single-cell amperometry. This technique monitors the release of serotonin from single granules in real-time with sub-millisecond temporal resolution. mDivi-1 exposure (10 μM) retarded each release event, resulting in a prolonged spike width of 23.00 ± 1.702 msec compared to the control value of 14.71 ± 1.194 msec. Although this concentration of mDivi-1 did not change the overall percentage of the fusion pore events or the amount of serotonin released through the fusion pore, it showed a distinct effect on the transition from stable fusion pore to maximal fusion pore dilation (% foot= 17.46 ± 1.809%, 9.464 ± 2.014% for control and mDivi-1 conditions, respectively). Evaluation of fluorescein-dextran incorporation into activated platelets by fluorescence microscopy enabled visualization of fusion pore dynamics and confirmed the effect of mDivi-1 on fusion pore expansion. To assess whether Drp-1 participates in platelet function in vivo, we determined the effect of mDivi-1 on thrombus formation following laser-induced injury of mouse cremaster arterioles. mDivi-1 inhibited platelet accumulation at the site of vascular injury by 74%. In contrast, mDivi-1 had no significant effect on fibrin formation under the same conditions. These results identify Drp-1 in platelets, demonstrate a role for Drp-1 in fusion pore dynamics, and indicate that pharmacological regulation of platelet fusion pore expansion can be used to control thrombus formation in vivo. Disclosures:No relevant conflicts of interest to declare.

Single-molecule transport across an individual biomimetic nuclear pore complex

Nuclear pore complexes regulate the selective exchange of RNA and proteins across the nuclear envelope in eukaryotic cells. Biomimetic strategies offer new opportunities to investigate this remarkable transport phenomenon. Here, we show selective transport of proteins across individual biomimetic nuclear pore complexes at the single-molecule level. Each biomimetic complex is constructed by covalently tethering either Nup98 or Nup153 (phenylalanine-glycine (FG) nucleoporins) to a solid-state nanopore. Individual translocation events are monitored using ionic current measurements with sub-millisecond temporal resolution. Transport receptors (Impβ) proceed with a dwell time of ∼2.5 ms for both Nup98- and Nup153-coated pores, whereas the passage of non-specific proteins is strongly inhibited with different degrees of selectivity. For pores up to ∼25 nm in diameter, Nups form a dense and low-conducting barrier, whereas they adopt a more open structure in larger pores. Our biomimetic nuclear pore complex provides a quantitative platform for studying nucleocytoplasmic transport phenomena at the single-molecule level in vitro.

Elementary Mechanisms of Force Generation

Elementary mechanisms of force generationAmin L.1, Shahapure R.1, Ercolini E.1,2, & Torre V.1,31International School for Advanced Studies (SISSA-ISAS), Trieste, Italy. 2 Cluster in Biomedicine (CBM), Area Science Park Basovizza, Trieste, Italy. 3Italian Institute of Technology, ISAS Unit, Italy.By using optical tweezers, we have characterized in details the mechanism of force generation in neuronal filopodia and lamellipodia with a nanometer sensitivity and sub-millisecond temporal resolution. Filopodia of dorsal root ganglia (DRG) neurons exert forces up to 2 pN, while those of hippocampal neurons a larger force up to 4-6 pN. When lamellipodia of DRG or hippocampal growth cones grow pushing a trapped bead, the autocorrelation of bead fluctuations decays with multiple time constants, while during Brownian fluctuations a single time constant of approximately 1 msec is observed. During push the power spectrum density of bead fluctuations is not fitted by a Lorentzian distribution and the energy content of low frequencies is higher than during Brownian fluctuations. Bead fluctuations along the three spatial components during push exhibit a higher cross-correlation than during Brownian fluctuations. Thus, during push the underlying dynamics is spatially and temporally correlated. Several algorithms show that during push, forward and backward jumps of 2-20 nm are detected. The net protrusion in a given time window is equal to the sum of all forward and backward jumps in the same window. These forward and backward jumps are identified as bursts of actin polymerization and depolymerization respectively and represent the elementary mechanisms of force generation.

Three-Dimensional Tracking of Single Fluorescent Particles with Submillisecond Temporal Resolution

Observing dynamics at the nanoscale requires submillisecond time resolution. Notably, in studying biological systems, three-dimensional (3D) trajectories of fluorescently labeled objects such as viruses or transport vesicles often need to be acquired with high temporal resolution. Here, we present a novel instrument that combines scanning-free multiplane detection at 3.2 kHz frame rate and single photon sensitivity with optimized beam-steering capabilities. This setup enables ultrafast 3D localization with submillisecond time resolution and nanometer localization precision. We demonstrate 3D tracking of single fluorescent particles at speeds of up to 150 nm/ms over several seconds and large volumes. By focused excitation of only the particle of interest, while avoiding confocal pinholes, the system realizes maximum detection efficiency at minimal laser irradiation. These features, combined with the avoidance of stage movement, provide high live-sample compatibility for future biomedical applications.

Functional localization of ion channel densities from calcium fluorescence

Single cells learn by tuning their synaptic conductances and redistributing their excitable machinery. To reveal its learning rules, one must therefore know how the cell remaps its ion channels in response to physiological stimuli. We exploit the recent ability to dynamically monitor cytosolic dye-buffered calcium, throughout rat hippocampal pyramidal cells in slice, with sub-millisecond temporal resolution and sub-micron spatial resolution [1] in the construction of a functional map of calcium and potassium channel densities. In the process we pose and solve [2] a number of inverse problems associated with converting dye recordings to current and voltage information through a series of experimental procedures: (1) Focal uncaging of cytosolic calcium in the presence of dye to determine dye kinetics and intracellular calcium extrusion rates, (2) Suprathreshold current injection at the soma to infer calcium current from calcium fluorescence using (1), (3) Recovery of the calcium channel density and voltage information from the calcium current in (2), (4) Recovery of various potassium channel densities through voltage information in (3) and the selective use of potassium channel blockers. We demonstrate the validity of this algorithm on synthetic data generated from channel densities and morphologies consistent with previous experimental results [3]. Finally, we explore the robustness of this algorithm to experimental error and noise.

Nanowire transistor arrays for mapping neural circuits in acute brain slices

Revealing the functional connectivity in natural neuronal networks is central to understanding circuits in the brain. Here, we show that silicon nanowire field-effect transistor (Si NWFET) arrays fabricated on transparent substrates can be reliably interfaced to acute brain slices. NWFET arrays were readily designed to record across a wide range of length scales, while the transparent device chips enabled imaging of individual cell bodies and identification of areas of healthy neurons at both upper and lower tissue surfaces. Simultaneous NWFET and patch clamp studies enabled unambiguous identification of action potential signals, with additional features detected at earlier times by the nanodevices. NWFET recording at different positions in the absence and presence of synaptic and ion-channel blockers enabled assignment of these features to presynaptic firing and postsynaptic depolarization from regions either close to somata or abundant in dendritic projections. In all cases, the NWFET signal amplitudes were from 0.3-3 mV. In contrast to conventional multielectrode array measurements, the small active surface of the NWFET devices, approximately 0.06 microm(2), provides highly localized multiplexed measurements of neuronal activities with demonstrated sub-millisecond temporal resolution and, significantly, better than 30 microm spatial resolution. In addition, multiplexed mapping with 2D NWFET arrays revealed spatially heterogeneous functional connectivity in the olfactory cortex with a resolution surpassing substantially previous electrical recording techniques. Our demonstration of simultaneous high temporal and spatial resolution recording, as well as mapping of functional connectivity, suggest that NWFETs can become a powerful platform for studying neural circuits in the brain.

An Address-Event Fall Detector for Assisted Living Applications

In this paper, we describe an address-event vision system designed to detect accidental falls in elderly home care applications. The system raises an alarm when a fall hazard is detected. We use an asynchronous temporal contrast vision sensor which features sub-millisecond temporal resolution. The sensor reports a fall at ten times higher temporal resolution than a frame-based camera and shows 84% higher bandwidth efficiency as it transmits fall events. A lightweight algorithm computes an instantaneous motion vector and reports fall events. We are able to distinguish fall events from normal human behavior, such as walking, crouching down, and sitting down. Our system is robust to the monitored person's spatial position in a room and presence of pets.

Using voltage‐sensitive dye recording to image the functional development of neuronal circuits in vertebrate embryos

Recent developments in the design of voltage-sensitive dyes and of recording apparatuses for detecting voltage-dependent changes in the optical properties of such dyes have established voltage-sensitive dye recording as an important technique for assessing the functional development of neuronal circuits in the brain and spinal cord. Here we discuss general technical issues regarding the recording of voltage-sensitive dye signals and describe studies that have utilized this approach to follow the development of sensory and sensorimotor circuits in the embryonic brain stem. Functional imaging through voltage-sensitive dye recording permits a noninvasive analysis of synaptic development and function at submillisecond temporal resolution in widely distributed circuits. These advantages are particularly valuable in assessing sensorimotor circuit development at early stages when neurons are small and synapses are fragile.

Action potential propagation imaged with high temporal resolution near-infrared video microscopy and polarized light

To identify the neural constituents responsible for generating polarized light changes, we created spatially resolved movies of propagating action potentials from stimulated lobster leg nerves using both reflection and transmission imaging modalities. Changes in light polarization are associated with membrane depolarization and provide sub-millisecond temporal resolution. Typically, signals are detected using light transmitted through tissue; however, because we eventually would like to apply polarization techniques in-vivo, reflected light is required. In transmission mode, the optical signal was largest throughout the center of the nerve, suggesting that most of the optical signal arose from the inner nerve bundle. In reflection mode, polarization changes were largest near the edges, suggesting that most of the optical signal arose from the outer sheath. In support of these observations, an optical model of the tissue showed that the outer sheath is more reflective while the inner nerve bundle is more transmissive. In order to apply these techniques in-vivo, we must consider that brain tissue does not have a regular orientation of processes as in the lobster nerve. We tested the effect of randomizing cell orientation by tying the nerve in an overhand knot prior to imaging, producing polarization changes that can be imaged even without regular cell orientations.

Magnetoencephalography in pediatric neuroimaging

AbstractNeural currents give rise to electroencephalogram (EEG) and magnetoencephalogram (MEG). MEG has selective sensitivity to tangential currents (from fissural cortex), and less distorted signals compared with EEG. A major goal of MEG is to determine the location and timing of cortical generators for event‐related responses, spontaneous brain oscillations or epileptiform activity. MEG provides a spatial accuracy of a few mm under optimal conditions, combined with an excellent submillisecond temporal resolution, which together enable spatiotemporal tracking of distributed neural activities, e.g. during cognitive tasks or epileptic discharges. While the present focus of pediatric MEG is on tailored epilepsy surgery, the complete noninvasiveness of MEG also provides unlimited possibilities to study the brain functions of healthy and developmentally deviant children.

Magnetoencephalography and magnetic source imaging in children.

Magnetoencephalography is a technique that detects the magnetic fields associated with the intracellular current flow within neurons, unlike electroencephalography, which measures extracellular volume currents. Superconducting quantum interference devices are used to amplify these very small magnetic field signals. Magnetic source imaging is the combination of functional data derived from magnetoencephalographic recordings coregistered with structural magnetic resonance imaging (MRI). The utility of magnetic source imaging lies in the combination of the submillisecond temporal resolution of magnetoencephalography with the precise anatomic images provided by magnetic resonance imaging. As such, magnetic source imaging is a useful tool for noninvasive localization of the epileptogenic zone in children who are candidates for epilepsy surgery. Similarly, using magnetoencephalographic recordings with evoked and event-related potentials, magnetic source imaging holds great promise as a noninvasive method for precise localization of somatosensory, motor, language, visual, and auditory cortex. Finally, magnetic source imaging is proving a valuable research tool in the investigation of epilepsy, head trauma, brain plasticity, and disorders of language, memory, cognition, and executive function in children.

Chemically‐Specific Probes for the Atomic Force Microscope

AbstractThe atomic force microscope (AFM) measures force and displacement with high sensitivity and submillisecond temporal resolution. By functionalizing the AFM probe with specific chemical groups or macromolecules it is possible to characterize the chemical and physical properties of single molecules on the nanometer scale. In this paper we discuss the key issues that must be addressed when designing and characterizing a successful immobilization chemistry, and describe the chemistry we developed to covalently immobilize oligonucleotides in a specific orientation.

Dynamic neuroimaging of brain function.

To fully characterize the brain processes underlying sensorimotor and cognitive function, the spatial distribution of active regions, their interconnected regions must be measured. We describe methods for imaging brain sources from surface-recorded EEG and magnetoencephalographic data, called electromagnetic source imaging (EMSI). EMSI provides brain source locations within the common framework of magnetic resonance (MR) images of brain anatomy. This allows integration of data from other functional brain imaging methods, like positron emission tomography and functional MR imaging, which can improve the accuracy of EMSI localization. EMSI also provides submillisecond temporal resolution of the dynamic processes within brain systems. Examples are given of applications to visual perceptual and attentional studies.

High-speed Rotation and Speed Stability of the Sodium-driven Flagellar Motor in Vibrio alginolyticus

The Na +-driven flagellar motor in Vibrio alginolyticusrotates very fast. Rotation of a single flagellum on a stuck cell was measured by laser dark-field microscopy with submillisecond temporal resolution. The rotation rate increased with increasing external concentration of NaCl, and reached 1000 r.p.s. at 300 nM NaCl. The Na +influx through the motor should determine the rotation period (τ) and affect the speed stability. Fluctuation of the rotation period was analyzed at various rotation rates (from ∼50 r.p.s. to ∼1000 r.p.s.), which were changed by changing the external concentration of NaCl and the addition of a protonophore or a specific inhibitor. At high rotation rates (over 400 r.p.s), the observed rotation was stable, and the standard deviation of τ (σ τ) ranged from 7% to 16% of the average rotation period (<τ>). At low rotation rates (under 100 r.p.s.), the rotation period tended to fluctuate, and the distributions of τ were non-Gaussian. The value of σ τranged from 10 to 30% of<τ>. However, the observed minimum value of σ τat various rotation rates was approximately equal to the calculated standard deviation due to the rotational diffusion of the flagellar filament. These results suggest that the torque was stably generated at various Na +influxes through the motor. We observed large fluctuations that cannot be explained by rotational diffusion. We discuss the factors that induce the large fluctuation. f2 f2 Professor Yasou Imae died suddenly of a cerebral haemorrhage on July 2nd 1993. This article is dedicated to him. Abbreviations used: LDM, laser dark-field microscopy; CCCP, carbonylcyanide m-chlorophenylhydrazone; r.p.s., revolutions per second.

Combination of multichannel detection and fast time response in a multichord spectrometer

A high resolution spectrometer has been used to measure simultaneously plasma impurity ion temperatures and rotational velocities, with submillisecond temporal resolution. The dual requirements of detailed spectral detection and fast time response are satisfied by having two detection systems viewing the same dispersed spectrum. The first is a multichannel system (e.g., a two-dimensional charge coupled device camera, for multichord detection) and the other an assembly comprising of three photomultipliers. A matched double spectrometer (f=1.26 m) is used to disperse the spectrum and giving an overall inverse dispersion of 0.04 nm/mm. The dispersive power is about 20× greater than the more conventional systems and has a corresponding increase in etendu, or light throughput, for the same resolution. The combination has been successfully used for magnetohydrodynamics mode locking experiments on COMPASS-C tokamak where velocity shifts of ∼30 km−1 were measured to an accuracy of ∼1 km s−1 with a 40 μs time resolution.

Mechanical response of frog saccular hair bundles to the aminoglycoside block of mechanoelectrical transduction.

1. Deflections of the mechanosensory hair bundles on frog saccular hair cells were measured interferometrically, with submillisecond temporal and submicrometer spatial resolution, and with subnanometer displacement sensitivity. 2. The direction of the initial bundle deflection (toward the taller stereocilia) in response to a sudden application of aminoglycoside antibiotics shows that the mechanosensory channels are blocked in their mechanically open state. 3. The magnitude of the initial deflection is consistent with published data on the gating swing as derived from the gating compliance. 4. A delayed relaxation and frequently a reversal of the initial deflection were observed and are attributed to the previously reported mechanical adaptation mechanism, which is at least partially controlled by the influx of Ca2+ through the transduction channels. 5. Increases of low-frequency spontaneous motion were found at intermediate blocker concentrations. They can be well accounted for by the fluctuating force exerted on the bundle by the random binding and unbinding of blocker molecules. 6. The mechanical response of the hair bundle to aminoglycosides may be related to their acute and specific ototoxicity.

How to sample the entire length of a single muscle fiber quick-frozen after electrical point stimulation for high resolution EM

We have developed a model that allows the quick-freezing at known time intervals following electrical field stimulation of a single, intact frog skeletal muscle fiber isolated by sharp dissection. The preparation is used for studying high resolution morphology by freeze-substitution and freeze-fracture and for electron probe x-ray microanlysis of sudden calcium displacement from intracellular stores in freeze-dried cryosections, all in the same fiber. We now show the feasibility and instrumentation of new methodology for stimulating a single, intact skeletal muscle fiber at a point resulting in the propagation of an action potential, followed by quick-freezing with sub-millisecond temporal resolution after electrical stimulation, followed by multiple sampling of the frozen muscle fiber for freeze-substitution, freeze-fracture (not shown) and cryosectionmg. This model, at once serving as its own control and obviating consideration of variances between different fibers, frogs etc., is useful to investigate structural and topochemical alterations occurring in the wake of an action potential.