Interpulse Delay Research Articles

Extracting Scalar Couplings From Complex 1H NMR Spectra Using a Simple 2D J-Resolved Sequence.

Measurement of scalar couplings between protons is a very challenging task because of complex multiplet patterns and severe overlapping of these multiplets in congested 1D spectra. Numerous 2D J-resolved sequences now exist that utilize either the Zangger-Sterk or PSYCHE or z-filter elements along with selective refocusing and pure-shift schemes to generate high-resolution phase-sensitive spectra with simple doublets in dimension. Herein, we present a 2D J-resolved sequence that employs a simple element consisting of hard pulses and inter-pulse delays to generate phase-sensitive spectra. This simple element in combination with selective refocusing eliminates all the undesired components including the intense axial peaks, thus provides clean 2D J-resolved spectra with signals of only two targeted protons with simple doublets in dimension and full multiplets of target protons in dimension. This high selectivity thus obviates the need for extra filtering elements and pure-shift acquisition schemes that are integrated into existing sequences to facilitate coupling measurements in overcrowded signals. It is therefore anticipated that this sequence, with the ease of implementation and ability to extract coupling values from highly congested spectra, should turn out an important tool for structural and conformational analyses in chemical and biological studies.

Ultrafast dynamics and internal processing mechanism of silica glass under double-pulse femtosecond laser irradiation

Femtosecond laser-induced plasma filaments have potential for various applications including attosecond physics, spectroscopy, and microprocessing. However, the use of plasma filaments to generate high-aspect-ratio internal modifications remains low-efficiency. Here, we experimentally demonstrated high-efficiency internal processing using plasma filaments induced by a double-pulse femtosecond laser. The processing mechanism was revealed through an investigation of the ultrafast dynamics of plasma filaments in experiments and simulations. We found that the excitation region of the first pulse (P1) exerted a temporal effect on the propagation and absorption of the second pulse (P2) due to the evolution of excited electrons, thus resulting in different processing characterizations. At a smaller inter-pulse delay (IPD), electrons and self-trapped excitons induced by P1 improved the absorption of P2 in the shallow region. Consequently, the main excitation regions of P1 and P2 were separated, resulting in a lower density of energy deposition and weak modifications. Whereas, at a larger IPD, P2 penetrated a deeper region with the relaxation of electrons and excitons induced by P1, leading to a better overlap of excitation regions between P2 and P1, thus improving the density of energy deposition and achieving efficient microprocessing. Besides, at an infinite IPD, P2 behaved like P1, but no modification was obtained owing to the complete energy diffusion of P1. Therefore, controlling the electron dynamic and energy diffusion contributes to the improvement of modification efficiency. Furthermore, the distribution of electron densities on the cross section was estimated to precisely analyze the microprocessing. These results are expected to aid in a better understanding of the interaction mechanism between dielectrics and intense ultrafast lasers and be useful for microprocessing applications.

Enhancing analytical merits of laser-induced breakdown spectroscopy of hydrogen isotopes using an orthogonal double-pulsing scheme

Accurate detection and quantification of hydrogen isotopes in solid materials are vital for diverse applications, including fusion energy, hydrogen storage, and tritium production. Laser-induced breakdown spectroscopy (LIBS) is a well-established, rapid, standoff method for this purpose, but it faces challenges related to the analytical merits required for isotopic analyses. In this study, we enhance the analytical and detection capabilities of traditional single-pulse LIBS by implementing an orthogonal double pulsing approach, focusing on the analysis of a range of 2H concentrations in Zircaloy-4 substrates (acting as a proxy for 3H). The double-pulse experiments employed an orthogonal re-heating configuration with two nanosecond Nd:YAG lasers. We systematically evaluated critical parameters affecting the signal intensity in double-pulse LIBS, including interpulse delay, ambient gas pressure, and heating laser energy. Our results demonstrate that employing an orthogonal double-pulse scheme significantly enhances 2Hα emission while minimizing line broadening and self-absorption, ultimately improving the technique's analytical capabilities.

Pulse interaction induced systematic errors in dual comb spectroscopy

Systematic errors are observed in dual comb spectroscopy when pulses from the two sources travel in a common fiber before interrogating the sample of interest. When sounding a molecular gas, these errors distort both the line shapes and retrieved concentrations. Simulations of dual comb interferograms based on a generalized nonlinear Schrodinger equation highlight two processes for these systematic errors. Self-phase modulation changes the spectral content of the field interrogating the molecular response but affects the recorded spectral baseline and absorption features differently, leading to line intensity errors. Cross-phase modulation modifies the relative inter-pulse delay, thus introducing interferogram sampling errors and creating a characteristic asymmetric distortion on spectral lines. Simulations capture the shape and amplitude of experimental errors which are around 0.1% on spectral transmittance residuals for 10 mW of total average power in 10 meters of common fiber, scaling up to above 0.6% for 20 mW and 60 m.

Dipolar Order Induced Electron Spin Hyperpolarization.

The structure of coupled electron spin systems is of fundamental interest to many applications, including dynamic nuclear polarization (DNP), enhanced nuclear magnetic resonance (NMR), the generation of electron spin qubits for quantum information science (QIS), and quantitative studies of paramagnetic systems by electron paramagnetic resonance (EPR). However, the characterization of electron spin coupling networks is nontrivial, especially at high magnetic fields. This study focuses on a system containing high concentrations of trityl radicals that give rise to a DNP enhancement profile of 1H NMR characteristic of the presence of electron spin clusters. When this system is subject to selective microwave saturation through pump-probe ELectron DOuble Resonance (ELDOR) experiments, electron spin hyperpolarization is observed. We show that the generation of an out-of-equilibrium longitudinal dipolar order is responsible for the transient hyperpolarization of electron spins. Notably, the coupled electron spin system needs to form an AX-like system (where the difference in the Zeeman interactions of two spins is larger than their coupling interaction) such that selective microwave irradiation can generate signatures of electron spin hyperpolarization. We show that the extent of dipolar order, as manifested in the extent of electron spin hyperpolarization generated, can be altered by tuning the pump or probe pulse length, or the interpulse delay in ELDOR experiments that change the efficiency to generate or readout longitudinal dipolar order. Pump-probe ELDOR with selective saturation is an effective means for characterizing coupled electron spins forming AX-type spin systems that are foundational for DNP and quantum sensing.

Frame change technique for phase transient cancellation

The precise control of complex quantum mechanical systems can unlock applications ranging from quantum simulation to quantum computation. Controlling strongly interacting many-body systems often relies on Floquet Hamiltonian engineering that is achieved by fast switching between Hamiltonian primitives via external control. For example, in our solid-state NMR system, we perform quantum simulation by modulating the natural Hamiltonian with control pulses. As the Floquet heating errors scale with the interpulse delay, δt, it is favorable to keep δt as short as possible, forcing our control pulses to be short duration and high power. Additionally, high-power pulses help to minimize undesirable evolution from occurring during the duration of the pulse. However, such pulses introduce an appreciable phase-transient control error, a form of unitary error. In this work, we detail our ability to diagnose the error, calibrate its magnitude, and correct it for π/2-pulses of arbitrary phase. We demonstrate the improvements gained by correcting for the phase transient error, using a method which we call the “frame-change technique”, in a variety of experimental settings of interest. Given that the correction mechanism adds no real control overhead, we recommend that any resonance probe be checked for these phase transient control errors, and correct them using the frame-change technique.

Parahydrogen-Induced Polarization of 14N Nuclei.

Hyperpolarization techniques provide a dramatic increase in sensitivity of nuclear magnetic resonance spectroscopy and imaging. In spite of the outstanding progress in solution-state hyperpolarization of spin-1/2 nuclei, hyperpolarization of quadrupolar nuclei remains challenging. Here, hyperpolarization of quadrupolar 14N nuclei with natural isotopic abundance of >99 % is demonstrated. This is achieved via pairwise addition of parahydrogen to tetraalkylammonium salts with vinyl or allyl unsaturated moieties followed by a subsequent polarization transfer from 1H to 14N nuclei at high magnetic field using PH-INEPT or PH-INEPT+ radiofrequency pulse sequence. Catalyst screening identified water-soluble rhodium complex [Rh(P(m-C6H4SO3Na)3)3Cl] as the most efficient catalyst for hyperpolarization of the substrates under study, providing up to 1.3 % and up to 6.6 % 1H polarization in the cases of vinyl and allyl precursors, respectively. The performance of PH-INEPT and PH-INEPT+ pulse sequences was optimized with respect to interpulse delays, and the resultant experimental dependences were in good agreement with simulations. As a result, 14N NMR signal enhancement of up to 760-fold at 7.05 T (corresponding to 0.15 % 14N polarization) was obtained.

Two-photon excitation two-dimensional fluorescence spectroscopy (2PE-2DFS) of the fluorescent nucleobase 6-MI.

Base stacking is fundamentally important to the stability of double-stranded DNA. However, few experiments can directly probe the local conformations and conformational fluctuations of the DNA bases. Here we report a new spectroscopic approach to study the local conformations of DNA bases using the UV-absorbing fluorescent guanine analogue, 6-methyl isoxanthopterin (6-MI), which can be used as a site-specific probe to label DNA. In these experiments, we apply a two-photon excitation (2PE) approach to two-dimensional fluorescence spectroscopy (2DFS), which is a fluorescence-detected nonlinear Fourier transform spectroscopy. In 2DFS, a repeating sequence of four collinear laser pulses (with center wavelength ~ 675 nm and relative phases swept at radio frequencies) is used to excite the lowest energy electronic-vibrational (vibronic) transitions of 6-MI (with center wavelength ~ 340 nm). The ensuing low flux fluorescence is phase-synchronously detected at the level of individual photons and as a function of inter-pulse delay. The 2PE transition pathways that give rise to electronically excited state populations include optical coherences between electronic ground and excited states and non-resonant (one-photon-excited) virtual states. Our results indicate that 2PE-2DFS experiments can provide information about the electronic-vibrational spectrum of the 6-MI monomer, in addition to the conformation-dependent exciton coupling between adjacent 6-MI monomers within a (6-MI)2 dimer. In principle, this approach can be used to determine the local base-stacking conformations of (6-MI)2 dimer-substituted DNA constructs.

Study of spectral lines behaviour with a double pulse 1064–1064 nm laser-induced breakdown spectroscopy system at the direct analysis of algae on the filter

The nickel and zinc contamination of algae on a cellulose filter was studied with double pulse 1064–1064 nm laser-induced breakdown spectroscopy (LIBS). Samples of the green alga Desmodesmus subspicatus were contaminated with zinc and nickel. The surface concentrations on the filter calculated from the reference Inductively Coupled Plasma Mass Spectrometry analysis were 25 and 64 ng cm−2 for nickel and zinc respectively. The algae are resting on the filter after drying and a double adhesive tape was used to fix the sample on the microscopic glass. During optimization method a compromise of 23–30 mJ pulse energies were set for maintaining a detectable or highest signal intensity of the analyte and not destroying the filter. Best conditions for maximum analyte signal and minimum deviation between the spectral behaviour of the analyte and the compared line of Na I, K I, Mg I, Mg II, Mn I, Mn II, Ca I, Ca II, and C I was 700 ns gate delay and 0 s interpulse delay. The deviation was rather more influenced with the signal-to-noise ratio of the analyte line than the closeness of the upper-level energies of the compared lines. The filter without algae showed systematically lower electron number density and total emissivity by units of percents than the filter with algae.

Using double pulse laser ablation in air to enhance the strength of laser-driven shocks

In the process of multi-pulse laser ablation, inter-pulse delay time, Δt, is known to be an important parameter for maximizing ablation efficiency as well as impulse imparted to the target. In this work, using photon Doppler velocimetry, we show that for single pairs of colinear pulses (1064 nm, 8 ns, ∼ 60 J cm-2 per pulse) in air, the peak free surface velocity of the back surface of an aluminum target (125 µm thick) is increased, by a factor of nearly 3, when Δt = 10 microseconds, compared with both pulses arriving simultaneously (Δt = 0). Fast imaging of the ablation process suggests this enhancement is due to rarefaction of the contiguous air in the passage of the leading shock produced by ablation, which then in turn allows a larger fraction of the energy of the second pulse to reach the target surface. This interpretation is strengthened by additional experiments in which the two pulses do not overlap on the target surface, but the shock strength is nevertheless enhanced. Given a fixed energy budget this work suggests a prescription for maximizing laser-driven shock strength by judicious choice of inter-pulse delay.

Sidebands in CEST MR-How to recognize and avoid them.

Clinical scanners require pulsed CEST sequences to maintain amplifier and specific absorption rate limits. During off-resonant RF irradiation and interpulse delay, the magnetization can accumulate specific relative phases within the pulse train. In this work, we show that these phases are important to consider, as they can lead to unexpected artifacts when no interpulse gradient spoiling is performed during the saturation train. We investigated sideband artifacts using a CEST-3D snapshot gradient-echo sequence at 3 T. Initially, Bloch-McConnell simulations were carried out with Pulseq-CEST, while measurements were performed in vitro and in vivo. Sidebands can be hidden in Z-spectra, and their structure becomes clearly visible only at high sampling. Sidebands are further influenced by B0 inhomogeneities and the RF phase cycling within the pulse train. In vivo, sidebands are mostly visible in liquid compartments such as CSF. Multi-pulse sidebands can be suppressed by interpulse gradient spoiling. We provide new insights into sidebands occurring in pulsed CEST experiments and show that, similar as in imaging sequences, gradient and RF spoiling play an important role. Gradient spoiling avoids misinterpretations of sidebands as CEST effects especially in liquid environments including pathological tissue or for CEST resonances close to water. It is recommended to simulate pulsed CEST sequences in advance to avoid artifacts.

Detection of gold alloy elements by target-enhanced orthogonal triple-pulse laser-induced breakdown spectroscopy with improved sensitivity and minimal sample ablation

In order to realize more sensitive elemental analysis of precious samples under minimal sample ablation, target-enhanced orthogonal triple-pulse laser-induced breakdown spectroscopy (TEOTP-LIBS) was first developed. In this technique, a pre-ablation laser with adequate interpulse delay was introduced in reheating target-enhanced orthogonal double-pulse laser-induced breakdown spectroscopy (TEODP-LIBS); the pre-ablation laser was collinearly propagated with the reheating laser and both interacted with a solid KHCO3 target. 5–7 folds signal enhancement caused by the pre-ablation laser could be observed. A maximum of 256 folds total signal enhancement has been achieved for Au I 267.595 nm transition line while analyzing an 18 K gold sample, if comparing TEOTP-LIBS with single-pulse LIBS using equal pulse energy for the ablation laser. 5.4–11.3 folds total improvement on the signal-to-noise ratio could be observed. The mechanism of the signal enhancement contributed by the pre-ablation laser was also discussed. It was demonstrated that TEOTP-LIBS was able to provide a better solution on the sensitive elemental analysis of precious samples under minimal sample ablation than TEODP-LIBS.

Laser-Induced Breakdown Spectroscopy Optimization Using Response Surface Methodology

Laser-induced breakdown spectroscopy (LIBS) is a modern analytical technique that is capable of fast, multi-elemental, lowcost and environmental friendly analysis, which does not require complex sample preparation. Albeit its potential, LIBS analysis still presents many limitations in terms of sensitivity and reproducibility, especially when analyzing complex matrices such as of sediments and soil samples. In order to reduce these matrix related effects, it is highly recommended that the system temporal parameters, which are responsible for controlling plasma evolution and signal collection, are optimized beforehand. In this work, we proposed the design of experiments (DOE) tool – specifically, the response surface methodology (RSM) – as an approach to optimize LIBS’s most important parameters (delay-time, interpulse delay, gate width and accumulated pulse). Signal-to-noise ratio (SNR) of the emission lines for Zn, Cd, Mg, Al, Ni, Cu, Ca, Cr, Sr, Fe were the response variable assessed during the procedure. The results showed that the RSM was an effective optimization tool for LIBS parameters and the final condition improved SNR ratios by up to a 48 ratio, when comparing to the not-optimal conditions.

Toward Large Ablations With Single-Needle High-Frequency Irreversible Electroporation in Vivo.

Irreversible electroporation (IRE) is a minimally thermal tissue ablation modality used to treat solid tumors adjacent to critical structures. Widespread clinical adoption of IRE has been limited due to complicated anesthetic management requirements and technical demands associated with placing multiple needle electrodes in anatomically challenging environments. High-frequency irreversible electroporation (H-FIRE) delivered using a novel single-insertion bipolar probe system could potentially overcome these limitations, but ablation volumes have remained small using this approach. While H-FIRE is minimally thermal in mode of action, high voltages or multiple pulse trains can lead to unwanted Joule heating. In this work, we improve the H-FIRE waveform design to increase the safe operating voltage using a single-insertion bipolar probe before electrical arcing occurs. By uniformly increasing interphase ( d1) and interpulse ( d2) delays, we achieved higher maximum operating voltages for all pulse lengths. Additionally, increasing pulse length led to higher operating voltages up to a certain delay length ( ∼25 μs), after which shorter pulses enabled higher voltages. We then delivered novel H-FIRE waveforms via an actively cooled single-insertion bipolar probe in swine liver in vivo to determine the upper limits to ablation volume possible using a single-needle H-FIRE device. Ablations up to 4.62 ± 0.12cm x 1.83 ± 0.05cm were generated in 5 minutes without a requirement for cardiac synchronization during treatment. Ablations were minimally thermal, easily visualized with ultrasound, and stimulated an immune response 24 hours post H-FIRE delivery. These data suggest H-FIRE can rapidly produce clinically relevant, minimally thermal ablations with a more user-friendly electrode design.

The Effects of Interphase and Interpulse Delays and Pulse Widths on Induced Muscle Contractions, Pain and Therapeutic Efficacy in Electroporation-Based Therapies.

Electroporation is used in medicine for drug and gene delivery, and as a nonthermal ablation method in tumor treatment and cardiac ablation. Electroporation involves delivering high-voltage electric pulses to target tissue; however, this can cause effects beyond the intended target tissue like nerve stimulation, muscle contractions and pain, requiring use of sedatives or anesthetics. It was previously shown that adjusting pulse parameters may mitigate some of these effects, but not how these adjustments would affect electroporation's efficacy. We investigated the effect of varying pulse parameters such as interphase and interpulse delay while keeping the duration and number of pulses constant on nerve stimulation, muscle contraction and assessing pain and electroporation efficacy, conducting experiments on human volunteers, tissue samples and cell lines in vitro. Our results show that using specific pulse parameters, particularly short high-frequency biphasic pulses with short interphase and long interpulse delays, reduces muscle contractions and pain sensations in healthy individuals. Higher stimulation thresholds were also observed in experiments on isolated swine phrenic nerves and human esophagus tissues. However, changes in the interphase and interpulse delays did not affect the cell permeability and survival, suggesting that modifying the pulse parameters could minimize adverse effects while preserving therapeutic goals in electroporation.

Plasma dynamics and chlorine emission characteristics on cement pastes using collinear dual-pulse laser-induced breakdown spectroscopy

The ingress of chloride can lead to the pitting corrosion of rebars, causing severe damage to concrete structures during service. Laser-induced breakdown spectroscopy (LIBS) has been demonstrated as an in-situ and reliable technique that can present cement distribution in concrete profiles, where the chlorine concentration is severely limited in the China standard. Dual-pulse LIBS has frequently been reported to enhance plasma emission. The evolution of the induced plasma generated from cement pastes is studied using an optical diagnostic system coupled with fast photography, shadowgraph, and optical emission spectroscopy, which can simultaneously record time-resolved physical information, including plasma morphology, shockwave propagation, and spatially resolved emission spectra in both single-pulse and dual-pulse configurations. The transition of the action mechanism of the second laser pulse from “initial plasma reheating” to “ablation enhancement” is demonstrated, which is responsible for the change in the plasma morphology from hemispherical to umbrella-shaped. It is observed that the new shockwave formed inside the initial shockwave is produced by the secondary ablation, which propagates faster, and shockwave fusion occurs when it approaches the initial shockwave. The spatially resolved Cl I emission at 837.59 nm is acquired, and the signal-to-background ratio (SBR) closely follows the profile of plasma temperature. The maximum SBR (> 3.0) can be accessed in the region where the plasma temperature reaches its peak (> 9200 K). Under the excitation of dual pulse with an inter-pulse delay of 200 ns, the Cl I emission can be clearly identified in the sample with a chlorine concentration of 0.252 wt% close to the critical value (0.2 wt% in the China standard), and a calibration curve is constructed with limit of detection (LOD) estimated to be 1930 ppm (dual pulse) versus 2550 ppm (single pulse).

Ablation depth enhancement on a copper surface using a dual-color double-pulse femtosecond laser.

We investigate the femtosecond laser ablation of copper with a dual-color double-pulse femtosecond laser at the wavelengths of 515 nm and 1030 nm. By properly choosing the energy of the 515 nm pulse, the optical properties such as surface reflectivity and absorption coefficient on copper surface can be modified to increase the absorption of the subsequent 1030 nm pulse. The ablation depth of dual-color double-pulse laser is at least 50% higher than the total ablation depth of both the 515 nm and 1030 nm pulses, provided that the inter-pulse delay of the double-pulse laser is within the electron-phonon coupling time. The ablation depth enhancement on a copper surface using a dual-color double-pulse femtosecond laser is of significant interest for scientific research and industrial application.

Resolving Nonlinear Recombination Dynamics in Semiconductors via Ultrafast Excitation Correlation Spectroscopy: Photoluminescence versus Photocurrent Detection.

We explore the application of excitation correlation spectroscopy to detect nonlinear photophysical dynamics in two distinct semiconductor classes through time-integrated photoluminescence and photocurrent measurements. In this experiment, two variably delayed femtosecond pulses excite the semiconductor, and the time-integrated photoluminescence or photocurrent component arising from the nonlinear dynamics of the populations induced by each pulse is measured as a function of inter-pulse delay by phase-sensitive detection with a lock-in amplifier. We focus on two limiting materials systems with contrasting optical properties: a prototypical lead-halide perovskite (LHP) solar cell, in which primary photoexcitations are charge photocarriers, and a single-component organic-semiconductor diode, which features Frenkel excitons as primary photoexcitations. The photoexcitation dynamics perceived by the two detection schemes in these contrasting systems are distinct. Nonlinear-dynamic contributions in the photoluminescence detection scheme arise from contributions to radiative recombination in both materials systems, while photocurrent arises directly in the LHP but indirectly following exciton dissociation in the organic system. Consequently, the basic photophysics of the two systems are reflected differently when comparing measurements with the two detection schemes. Our results indicate that photoluminescence detection in the LHP system provides valuable information about trap-assisted and Auger recombination processes, but that these processes are convoluted in a nontrivial way in the photocurrent response and are therefore difficult to differentiate. In contrast, the organic-semiconductor system exhibits more directly correlated responses in the nonlinear photoluminescence and photocurrent measurements, as charge carriers are secondary excitations only generated through exciton dissociation processes. We propose that bimolecular annihilation pathways mainly contribute to the generation of charge carriers in single-component organic semiconductor devices. Overall, our work highlights the utility of excitation correlation spectroscopy in modern semiconductor materials research, particularly in the analysis of nonlinear photophysical processes, which are deterministic for their electronic and optical properties.

Emission Enhancement in fs + ns Dual-Pulse LIBS of Cu

Femtosecond (fs) and nanosecond (ns) laser pulses have their own advantages and disadvantages in laser-induced breakdown spectroscopy (LIBS). This paper investigated fs + ns (FN) dual-pulse (DP) LIBS, utilizing the respective advantages of two laser pulses in LIBS. Compared to traditional single ns LIBS, applying a smaller energy fs pulse could effectively improve the LIBS emission. Firstly, this study discussed the spectra of FN DP LIBS with overlapping pulse time—that is, the FN DP inter-pulse delay (DID) was 0 μs. The results showed that the spectra were increased to three times that of a single ns LIBS. Subsequently, the DID between the two pulses was optimized. The results showed that as the DID between the two pulses increased, the spectral emission first increased and then decreased, ultimately remaining unchanged. The optimized DID was approximately 2 μs. Finally, using this optimized DID, the variation of spectral intensity with ns laser energy was discussed in DP LIBS. The spectral enhancement ratio increased from 3 with 0 μs DID to 6 with 2 μs DID. The investigation provides a reference in the application of FN DP LIBS element analysis.

Fluid flow influences ultrasound-assisted endothelial membrane permeabilization and calcium flux.

The local fluid dynamics experienced by circulating microbubbles vary across different anatomical sites, which can influence ultrasound-mediated therapeutic delivery efficacy. This study aimed to elucidate the effect of fluid flow rate in combination with repeated short-pulse ultrasound on microbubble-mediated endothelial cell permeabilization. Here, a seeded monolayer of human umbilical (HUVEC) or brain endothelial cells (HBEC-5i) was co-perfused with a solution of microbubbles and propidium iodide (PI) at either a flow rate of 5 or 30ml/min. Using an acoustically coupled inverted microscope, cells were exposed to 1MHz ultrasound with 20-cycle bursts, 1ms PRI, and 2s duration at a peak negative pressure of 305kPa to assess the role of flow rate on ultrasound-stimulated endothelial cell permeability, as well as Ca2+ modulation. In addition, the effect of inter-pulse delays (∆t=1s) on the resulting endothelial permeability was investigated. Our results demonstrate that under an identical acoustic stimulus, fast-flowing microbubbles resulted in a statistically significant increase in cell membrane permeability, at least by 2.3-fold, for both endothelial cells. Likewise, there was a substantial difference in intracellular Ca2+ levels between the two examined flow rates. In addition, multiple short pulses rather than a single pulse ultrasound, with an equal number of bursts, significantly elevated endothelial cell permeabilization, at least by 1.4-fold, in response to ultrasound-stimulated microbubbles. This study provides insights into the design of optimal, application-dependent pulsing schemes to improve the effectiveness of ultrasound-mediated local therapeutic delivery.