Millisecond Time Scale Research Articles

Super-resolution fluorescence microscopy for investigating bacterial cell biology.

Super-resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single-molecule resolution on a millisecond time scale. Here we review recent applications of super-resolution fluorescence microscopy with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of molecular machines in action.

Recent Advances in Neurotechnologies to Understand and Direct Specific Behavior of Live Animals

Neuroscience has traditionally faced difficulty in accessing and understanding the behavior of the brain at a single-cell level. Many of these challenges are due to the brain’s complexity; the brain has 100 billion neurons and well over 100 trillion connections and synapses, all operating at a millisecond timescale with functionally distinct circuits crossing over each other within microscale regions. However, starting with breakthroughs in the 2000s, such as fluorescent genetically-encoded calcium ion (Ca2+) indicators (GECIs)—where Ca2+ detection is used as a proxy of neural activity—and optogenetics—where neuron firing is controlled using light-sensitive ion channels in neurons—new advances in revolutionary neurotechnology have made it increasingly feasible for neuroscientists to understand how the brain operates on a cellular level. In the past few years, four distinct categories of neurotechnology advances have catalyzed this change in understanding, including chemogenic control, electrical recording and control, optogenetics-based control, and optogenetics recording and visualization.

Bright and sensitive red voltage indicators for imaging action potentials in brain slices and pancreatic islets.

Genetically encoded voltage indicators (GEVIs) allow the direct visualization of cellular membrane potential at the millisecond time scale. Among these, red-emitting GEVIs have been reported to support multichannel recordings and manipulation of cellular activities with reduced autofluorescence background. However, the limited sensitivity and dimness of existing red GEVIs have restricted their applications in neuroscience. Here, we report a pair of red-shifted opsin-based GEVIs, Cepheid1b and Cepheid1s, with improved dynamic range, brightness, and photostability. The improved dynamic range is achieved by a rational design to raise the electrochromic Förster resonance energy transfer efficiency, and the higher brightness and photostability are approached with separately engineered red fluorescent proteins. With Cepheid1 indicators, we recorded complex firings and subthreshold activities of neurons on acute brain slices and observed heterogeneity in the voltage‑calcium coupling on pancreatic islets. Overall, Cepheid1 indicators provide a strong tool to investigate excitable cells in various sophisticated biological systems.

Transsynaptic entrainment of cerebellar nuclear cells by alternating currents in a frequency dependent manner.

Transcranial alternating current stimulation (tACS) is a non-invasive neuromodulation technique that is being tested clinically for treatment of a variety of neural disorders. Animal studies investigating the underlying mechanisms of tACS are scarce, and nearly absent in the cerebellum. In the present study, we applied 10-400 Hz alternating currents (AC) to the cerebellar cortex in ketamine/xylazine anesthetized rats. The spiking activity of cerebellar nuclear (CN) cells was transsynaptically entrained to the frequency of AC stimulation in an intensity and frequency-dependent manner. Interestingly, there was a tuning curve for modulation where the frequencies in the midrange (100 and 150 Hz) were more effective, although the stimulation frequency for maximum modulation differed for each CN cell with slight dependence on the stimulation amplitude. CN spikes were entrained with latencies of a few milliseconds with respect to the AC stimulation cycle. These short latencies and that the transsynaptic modulation of the CN cells can occur at such high frequencies strongly suggests that PC simple spike synchrony at millisecond time scales is the underlying mechanism for CN cell entrainment. These results show that subthreshold AC stimulation can induce such PC spike synchrony without resorting to supra-threshold pulse stimulation for precise timing. Transsynaptic entrainment of deep CN cells via cortical stimulation could help keep stimulation currents within safety limits in tACS applications, allowing development of tACS as an alternative treatment to deep cerebellar stimulation. Our results also provide a possible explanation for human trials of cerebellar stimulation where the functional impacts of tACS were frequency dependent.

EEG microstates in early-to-middle childhood show associations with age, biological sex, and alpha power.

Electroencephalographic (EEG) microstates can provide a unique window into the temporal dynamics of large-scale brain networks across brief (millisecond) timescales. Here, we analysed fundamental temporal features of microstates extracted from the broadband EEG signal in a large (N = 139) cohort of children spanning early-to-middle childhood (4-12 years of age). Linear regression models were used to examine if participants' age and biological sex could predict the temporal parameters GEV, duration, coverage, and occurrence, for five microstate classes (A-E) across both eyes-closed and eyes-open resting-state recordings. We further explored associations between these microstate parameters and posterior alpha power after removal of the 1/f-like aperiodic signal. The microstates obtained from our neurodevelopmental EEG recordings broadly replicated the four canonical microstate classes (A to D) frequently reported in adults, with the addition of the more recently established microstate class E. Biological sex served as a significant predictor in the regression models for four of the five microstate classes (A, C, D, and E). In addition, duration and occurrence for microstate E were both found to be positively associated with age for the eyes-open recordings, while the temporal parameters of microstates C and E both exhibited associations with alpha band spectral power. Together, these findings highlight the influence of age and sex on large-scale functional brain networks during early-to-middle childhood, extending understanding of neural dynamics across this important period for brain development.

COVID-19 cases prediction with negative group delays digital function

The negative group delay (NGD) is an uncommon function enabling to propagate arbitrary waveform signals with time-advance behavior. The counterintuitive NGD function was initially experimented for anticipating typically fast and short duration electronic signals in micro- and milli-second time scale. The application of NGD function to large time scale signal attracts more and more the attention of data processing engineer. This paper aims to investigate on the ability of NGD function to predict time- dependent social data with someday time-advances. As practical case of study, an innovative application of NGD function for predicting disease cases is treated. The digital circuit theory enabling to understand the low-pass (LP) NGD canonical TF and the characterization approach is established. It is shown in which condition the first order difference equation represents a LP-NGD circuit. Then, the design method of typical LP-NGD predictor as numerical circuit is introduced in function of the expected time-advance. The NGD predictor time-variation property is theoretically initiated. The NGD time-advance varied from -7 days to -1/2 days is investigated with deterministic data prediction processing from 5-months bi- exponential waveform data. The predicted data with time-advance of about -4 days was confirmed by analytical computation and simulation. The LP-NGD digital predictor feasibility is validated with monthly COVID-19 randomly arbitrary data by computed and virtually tested results. It was investigated with sensitivity analysis that the prediction performance is better when the input signal is smoothed enough. As expected, prediction result showing very good correlation with input data is demonstrated.

Study and Optimization of Two-State Transient Currents at Millisecond Time Scales in MIS Tunnel Diodes

Study and Optimization of Two-State Transient Currents at Millisecond Time Scales in MIS Tunnel Diodes

Dilute oil-in-water emulsion initiates multiple holes formation during bubble bursting

The air bubble bursting dynamics involve intricate interfacial phenomena of millisecond timescale, which are of interest in many natural and industrial processes. The current study concerns bubble containing oil droplets with size comparable to bubble cap thickness. The assumption is that the dynamics of bubble bursting and aerosol formation will be modified as oil droplets interact with the air-water interface during drainage. Indeed, the experiments show that with the increase of the droplet spreading coefficient (S), the average film retraction speed decreases and the average film thickness increases at breakup. Notably, as S reaches 3.6 mN/m, a distinct multiple hole formation phenomenon has been observed for the first time during bubble bursting. The frequency of multiple hole formation increases with increasing S, and is as high as 22 holes per bursting event observed when S = 21.3 mN/m. The multiple holes evolve with time and form convoluted ligament structures that finally break up into much more aerosolized droplets. However, after normalization, all droplet size distribution is well predicated by a Gamma distribution.The results highlight the importance of the interfacial physiochemistry for the bubble cap rupture, regarding the wetting state between the oil droplets, air, water phase.

Rapid Kinetic Interactions of Sugar and Sugar Alcohol with Sweet Taste Receptors on Live Cells Using Stopped-Flow Spectroscopy.

The subjective measurement of the dynamic perception of sweetness is a problem in food science. Herein, the rapid interactions of sugars and sugar alcohols with sweet taste receptors on living cells on a millisecond timescale were studied via stopped-flow fluorescence spectroscopy. According to the rapid-kinetic parameters, sweeteners were divided into two groups. Sweeteners in group I disrupted the hydrogen bond network structure of water, and the apparent rate constant (kobs) was in the range of 0.45-0.6 s-1. Sweeteners in group II promoted the hydrogen bond formation of water, and the kobs was mostly in the range of 0.6-0.75 s-1. For most sweeteners, the kobs of cell responses was negatively correlated with the apparent specific volume of sweeteners. The differences in the cellular responses may be attributed to the disturbance in the water structure. Experimental results showed that the kinetic parameters of sweet cell responses reflected the dynamic perception of sweetness. Rapid kinetics, solution thermodynamic analysis, and water structure analysis enriched the physicochemical study of the sweetness mechanism and can be used to objectively evaluate the dynamic perception of sweetness.

Growth Anisotropy and Morphology Evolution of Line Defects in Monolayer MoS2 : Atomic-Level Observation, Large-Scale Statistics, and Mechanism Understanding.

Understanding the growth behavior and morphology evolution of defects in 2D transition metal dichalcogenides is significant for the performance tuning of nanoelectronic devices. Here, the low-voltage aberration-corrected transmission electron microscopy with an in situ heating holder and a fast frame rate camera to investigate the sulfur vacancy lines in monolayer MoS2 is applied. Vacancy concentration-dependent growth anisotropy is discovered, displaying first lengthening and then broadening of line defects as the vacancy densifies. With the temperature increase from 20 °C to 800 °C, the defect morphology evolves from a dense triangular network to an ultralong linear structure due to the temperature-sensitive vacancy migration process. Atomistic dynamics of line defect reconstruction on the millisecond time scale are also captured. Density functional theory calculations, Monte Carlo simulation, and configurational force analysis are implemented to understand the growth and reconstruction mechanisms at relevant time and length scales. Throughout the work, high-resolution imaging is closely combined with quantitative analysis of images involving thousands of atoms so that the atomic-level structure and the large-area statistical rules are obtained simultaneously. The work provides new ideas for balancing the accuracy and universality of discoveries in the TEM study and will be helpful to the controlled sculpture of nanomaterials.

Rapid droplet-based mixing for single-molecule spectroscopy.

Probing non-equilibrium dynamics with single-molecule spectroscopy is important for dissecting biomolecular mechanisms. However, existing microfluidic rapid-mixing systems for this purpose are incompatible with surface-adhesive biomolecules, exhibit undesirable flow dispersion and are often demanding to fabricate. Here we introduce droplet-based microfluidic mixing for single-molecule spectroscopy to overcome these limitations in a wide range of applications. We demonstrate its robust functionality with binding kinetics of even very surface-adhesive proteins on the millisecond timescale.

Assessing the energy release characteristics during the middle detonation reaction stage of aluminized explosives

Afterburning behind the detonation front of an aluminized explosive releases energy on the millisecond timescale, which prolong the release of detonation energy and the energy release at different stages also shows significant differences. However, at present, there are few effective methods for evaluating the energy release characteristics of the middle reaction stage of such explosives, which can have a duration of tens to hundreds of microseconds. The present work demonstrates an approach to assessing the mid-stage of an aluminized explosive detonation based on a water push test employing a high degree of confinement. In this method, the explosive is contained in a steel cylinder having one end closed that is installed at the bottom of a transparent water tank. Upon detonation, the gaseous products expand in one direction while forcing water ahead of them. The resulting underwater shock wave and the interface between the gas phase products and the water are tracked using an ultra-high-speed framing and streak camera. The shock wave velocity in water and the expansion work performed by the gaseous detonation products were calculated to assess the energy release characteristics of aluminized explosives such as CL-20 and RDX in the middle stage of the detonation reaction. During the middle stage of the detonation process of these aluminized explosives, the aluminum reaction reduced the attenuation of shock waves and increased the work performed by gas phase products. A higher aluminum content increased the energy output while the presence of oxidants slowed the energy release rate. This work demonstrates an effective means of evaluating the performance of aluminized explosives.

Order-of-magnitude SNR improvement for high-field EPR spectrometers via 3D printed quasi-optical sample holders.

Here, we present a rapidly prototyped, cost-efficient, and 3D printed quasi-optical sample holder for improving the signal-to-noise ratio (SNR) in modern, resonator-free, and high-field electron paramagnetic resonance (HFEPR) spectrometers. Such spectrometers typically operate in induction mode: The detected EPR ("cross-polar") signal is polarized orthogonal to the incident ("co-polar") radiation. The sample holder makes use of an adjustable sample positioner that allows for optimizing the sample position to maximize the 240-gigahertz magnetic field B1 and a rooftop mirror that allows for small rotations of the microwave polarization to maximize the cross-polar signal and minimize the co-polar background. When optimally tuned, the sample holder was able to improve co-polar isolation by ≳20 decibels, which is proven beneficial for maximizing the SNR in rapid-scan, pulsed, and continuous-wave EPR experiments. In rapid-scan mode, the improved SNR enabled the recording of entire EPR spectra of a narrow-line radical in millisecond time scales, which, in turn, enabled real-time monitoring of a sample's evolving line shape.

Photoswitching alters fluorescence readout of jGCaMP8 Ca2+ indicators tethered to Orai1 channels

We used electrophysiology and Ca2+ channel tethering to evaluate the performance of jGCaMP8 genetically encoded Ca2+ indicators (GECIs). Orai1 Ca2+ channel-jGCaMP8 fusions were transfected into HEK 293A cells and jGCaMP8 fluorescence responses recorded by simultaneous total internal reflection fluorescence microscopy and whole-cell patch clamp electrophysiology. Noninactivating currents from the Orai1 Y80E mutant provided a steady flux of Ca2+ controlled on a millisecond time scale by step changes in membrane potential. Test pulses to -100 mV produced Orai1 Y80E-jGCaMP8f fluorescence traces that unexpectedly declined by ~50% over 100 ms before reaching a stable plateau. Testing of Orai1-jGCaMP8f using unroofed cells further demonstrated that rapid and partial fluorescence inactivation is a property of the indicator itself, rather than channel function. Photoinactivation spontaneously recovered over 5 min in the dark, and recovery was accelerated in the absence of Ca2+. Mutational analysis of residues near the tripeptide fluorophore of jGCaMP8f pointed to a mechanism: Q69M/C70V greatly increased (~90%) photoinactivation, reminiscent of fluorescent protein fluorophore cis-trans photoswitching. Indeed, 405-nm illumination of jGCaMP8f or 8m/8s/6f led to immediate photorecovery, and simultaneous illumination with 405 and 488-nm light blocked photoinactivation. Subsequent mutagenesis produced a variant, V203Y, that lacks photoinactivation but largely preserves the desirable properties of jGCaMP8f. Our results point to caution in interpreting rapidly changing Ca2+ signals using jGCaMP8 and earlier series GECIs, suggest strategies to avoid photoswitching, and serve as a starting point to produce more photostable, and thus more accurate, GECI derivatives.

Ultrafast Switching of Interfacial Thermal Conductance.

Dynamical control of thermal transport at the nanoscale provides a time-domain strategy for optimizing thermal management in nanoelectronics, magnetic devices, and thermoelectric devices. However, the rate of change available for thermal switches and regulators is limited to millisecond time scales, calling for a faster modulation speed. Here, time-resolved X-ray diffraction measurements and thermal transport modeling reveal an ultrafast modulation of the interfacial thermal conductance of an FeRh/MgO heterostructure as a result of a structural phase transition driven by optical excitation. Within 90 ps after optical excitation, the interfacial thermal conductance is reduced by a factor of 5 and lasts for a few nanoseconds, in comparison to the value at the equilibrium FeRh/MgO interface. The experimental results combined with thermal transport calculations suggest that the reduced interfacial thermal conductance results from enhanced phonon scattering at the interface where the lattice experiences transient in-plane biaxial stress due to the structural phase transition of FeRh. Our results suggest that optically driven phase transitions can be utilized for ultrafast nanoscale thermal switches for device application.

Stability and Cooling of the C_{7}^{2-} Dianion.

We have studied the stability of the smallest long-lived all carbon molecular dianion (C_{7}^{2-}) in new time domains and with a single ion at a time using a cryogenic electrostatic ion-beam storage ring. We observe spontaneous electron emission from internally excited dianions on millisecond timescales and monitor the survival of single colder C_{7}^{2-} molecules on much longer timescales. We find that their intrinsic lifetime exceeds several minutes-6 orders of magnitude longer than established from earlier experiments on C_{7}^{2-}. This is consistent with our calculations of vertical electron detachment energies predicting one inherently stable isomer and one isomer which is stable or effectively stable behind a large Coulomb barrier for C_{7}^{2-}→C_{7}^{-}+e^{-} separation.

Cerebellar contributions across behavioural timescales: a review from the perspective of cerebro-cerebellar interactions.

Performing successful adaptive behaviour relies on our ability to process a wide range of temporal intervals with certain precision. Studies on the role of the cerebellum in temporal information processing have adopted the dogma that the cerebellum is involved in sub-second processing. However, emerging evidence shows that the cerebellum might be involved in suprasecond temporal processing as well. Here we review the reciprocal loops between cerebellum and cerebral cortex and provide a theoretical account of cerebro-cerebellar interactions with a focus on how cerebellar output can modulate cerebral processing during learning of complex sequences. Finally, we propose that while the ability of the cerebellum to support millisecond timescales might be intrinsic to cerebellar circuitry, the ability to support supra-second timescales might result from cerebellar interactions with other brain regions, such as the prefrontal cortex.

Rapid characterization of Maillard reaction products in heat-treated honey by nanoelectrospray ionization mass spectrometry.

Amadori rearrangement products (ARPs) and α-dicarbonyl compounds (α-DCs) are critical intermediates in the Maillard chemistry. The screening of artificially heated honey (AH) is currently based on chromatography-mass spectrometry, which is commonly accompanied with the longer pretreatment and detection time. Here, low-abundance ARPs were detected directly in high-sugar environment by nanoelectrospray ionization mass spectrometry (nanoESI-MS) coupled with borosilicate glass capillaries (O-tips). When O-tips were replaced by borosilicate theta capillaries (θ-tips), the microdroplets allowed the derivatization of α-DCs to be accomplished on the millisecond timescale, rather than hours in conventional protocols. The results indicated that two ARPs and α-DCs of m/z 235 were significantly up-regulated in AH. Meanwhile, the straightforward differentiation between naturally matured honey (NH) and AH was achieved by nanoESI-MS fingerprints combined with multivariate analysis. The method may provide a rapid characterization of Maillard reaction products (MRPs), which exhibits the great application potential in other complex food matrix.

Fast vs slow physical aging of a glass forming liquid.

Using electric fields to initiate the process of physical aging has facilitated measurements of structural recovery dynamics on the time scale of milliseconds. This, however, complicates the interesting comparison with aging processes due to a temperature jump, as these are significantly slower. This study takes a step toward comparing the results of field and temperature perturbations by providing data on field-induced structural recovery of vinyl ethylene carbonate at two different time scales: 1.0ms at 181K and 33s at 169K, i.e., 4.5 decades apart. It is found that structural recovery is a factor of two slower than structural relaxation in equilibrium, with the latter determined via dielectric relaxation in the limit of linear response. The relation between recovery and relaxation dynamics remains temperature invariant across the present experimental range.

Mechanistic Insights into Tyrosine Oxidation Revealed by Fast-Scan Cyclic Voltammetry

A precise understanding of opioid dynamics in the brain is lacking, largely because few analytical tools are available that are sufficiently sensitive and selective for directly monitoring these important neurochemicals in situ. Tyrosine is a key part of the opioid neuropeptides, and its redox activity can be exploited in their detection. However, a solid understanding of tyrosine electrochemistry on carbon electrodes is first required. It is known that, upon oxidation, tyrosine quickly undergoes chemical transformation to products that foul the electrode, compromising the electrochemical information. This issue can be avoided by using fast-scan cyclic voltammetry (FSCV) which enables collection of well-defined voltammograms on a millisecond timescale before appreciable accumulation of byproducts. In this work, the tyrosine oxidation reaction is characterized and electrode fouling is assessed when tyrosine is incorporated into short synthetic peptides containing other amino acids that are part of the opioid neuropeptides: Gly, Phe, Met, Leu, and Arg. Voltammetric data were obtained in phosphate buffered solution on carbon microelectrodes at scan rates up to 1 kV/s. The results demonstrate the dependence of the peak oxidation potential on solution pH, and identify possible reaction pathways and byproducts that foul the electrode. Peak oxidation potential, peak shape, and ultimately, sensitivity to tyrosine are dependent on the peptide sequence. NMR spectroscopy is used to relate the proximity of proton acceptor/donor functional groups to the electrochemical results in the context of proton-coupled electron transfer (PCET) theory. These fundamental studies are important for the electrochemical detection of important neuropeptides in the complex in vivo environment. Furthermore, a better understanding of the tunability of PCET reaction rates in electrochemical systems has broad implications for technology in a range of areas including artificial photosynthesis, solar energy conversion, and energy storage.