Genetic engineering biofilms in situ using ultrasound-mediated DNA delivery.

The ability to directly modify native and established biofilms has enormous potential in understanding microbial ecology and application of biofilm in 'real-world' systems. However, efficient genetic transformation of established biofilms at any scale remains challenging. In this study, we applied an ultrasound-mediated DNA delivery (UDD) technique to introduce plasmid to established non-competent biofilms in situ. Two different plasmids containing genes coding for superfolder green fluorescent protein (sfGFP) and the flavin synthesis pathway were introduced into established bacterial biofilms in microfluidic flow (transformation efficiency of 3.9 ± 0.3 × 10-7 cells in biofilm) and microbial fuel cells (MFCs), respectively, both employing UDD. Gene expression and functional effects of genetically modified bacterial biofilms were observed, where some cells in UDD-treated Pseudomonas putida UWC1 biofilms expressed sfGFP in flow cells and UDD-treated Shewanella oneidensis MR-1 biofilms generated significantly (P < 0.05) greater (61%) bioelectricity production (21.9 ± 1.2 µA cm-2 ) in MFC than a wild-type control group (~ 13.6 ± 1.6 µA cm-2 ). The effects of UDD were amplified in subsequent growth under selection pressure due to antibiotic resistance and metabolism enhancement. UDD-induced gene transfer on biofilms grown in both microbial flow cells and MFC systems was successfully demonstrated, with working volumes of 0.16 cm3 and 300 cm3 , respectively, demonstrating a significant scale-up in operating volume. This is the first study to report on a potentially scalable direct genetic engineering method for established non-competent biofilms, which can be exploited in enhancing their capability towards environmental, industrial and medical applications.

The influence of droplet concentration on phase change and inertial cavitation thresholds associated with acoustic droplet vaporization.

Acoustic droplet vaporization (ADV) is an important process that enables the theragnostic application of acoustically activated droplets, where the nucleation of inertial cavitation (IC) activity must be precisely controlled. This Letter describes threshold pressure measurements for ADV and acoustic emissions consistent with IC activity of lipid-shelled non-superheated perfluoropentane nanodroplets over a range of physiologically relevant concentrations at 1.1-MHz. Under the frequency investigated, results show that the thresholds were relatively independent of concentration for intermediate concentrations (105, 106, and 107 droplets/ml), thus indicating an optimal range of droplet concentrations for conducting threshold studies. For the highest concentration, the difference between the threshold for IC and the threshold for ADV was greatly reduced, suggesting that it might prove difficult to induce ADV without concomitant IC in applications that employ higher concentrations.

SVD-Based Separation of Stable and Inertial Cavitation Signals Applied to Passive Cavitation Mapping During HIFU.

Detection of inertial and stable cavitation is important for guiding high-intensity focused ultrasound (HIFU). Acoustic transducers can passively detect broadband noise from inertial cavitation and the scattering of HIFU harmonics from stable cavitation bubbles. Conventional approaches to cavitation noise diagnostics typically involve computing the Fourier transform of the time-domain noise signal, applying a custom comb filter to isolate the frequency components of interest, followed by an inverse Fourier transform. We present an alternative technique based on singular value decomposition (SVD) that efficiently separates the broadband emissions and HIFU harmonics. Spatiotemporally resolved cavitation detection was achieved using a 128-element, 5-MHz linear-array ultrasound imaging system operating in the receive mode at 15 frames/s. A 1.1-MHz transducer delivered HIFU to tissue-mimicking phantoms and excised liver tissue for a duration of 5 s. Beamformed radio frequency signals corresponding to each scan line in a frame were assembled into a matrix, and SVD was performed. Spectra of the singular vectors obtained from a tissue-mimicking gel phantom were analyzed by computing the peak ratio ( R ), defined as the ratio of the peak of its fifth-order polynomial fit and the maximum spectral peak. Singular vectors that produced an were classified as those representing stable cavitation, i.e., predominantly containing harmonics of HIFU. The projection of data onto this singular base reproduced stable cavitation signals. Similarly, singular vectors that produced an were classified as those predominantly containing broadband noise associated with inertial cavitation. These singular vectors were used to isolate the inertial cavitation signal. The R -value thresholds determined using gel data were then employed to analyze cavitation data obtained from bovine liver ex vivo. The SVD-based method faithfully reproduced the structural details in the spatiotemporal cavitation maps produced using the more cumbersome comb-filter approach with a maximum root-mean-squared error of 10%.

Photo- and Sono-Dynamic Therapy: A Review of Mechanisms and Considerations for Pharmacological Agents Used in Therapy Incorporating Light and Sound.

As irreplaceable energy sources of minimally invasive treatment, light and sound have, separately, laid solid foundations in their clinic applications. Constrained by the relatively shallow penetration depth of light, photodynamic therapy (PDT) typically involves involves superficial targets such as shallow seated skin conditions, head and neck cancers, eye disorders, early-stage cancer of esophagus, etc. For ultrasound-driven sonodynamic therapy (SDT), however, to various organs is facilitated by the superior... transmission and focusing ability of ultrasound in biological tissues, enabling multiple therapeutic applications including treating glioma, breast cancer, hematologic tumor and opening blood-brain-barrier (BBB). Considering the emergence of theranostics and precision therapy, these two classic energy sources and corresponding sensitizers are worth reevaluating. In this review, three typical therapies using light and sound as a trigger, PDT, SDT, and combined PDT and SDT are introduced. The therapeutic dynamics and current designs of pharmacological sensitizers involved in these therapies are presented. By introducing both the history of the field and the most up-to-date design strategies, this review provides a systemic summary on the development of PDT and SDT and fosters inspiration for researchers working on 'multi-modal' therapies involving light and sound.

HIFU-induced changes in optical scattering and absorption of tissue over nine orders of thermal dose.

The optical properties of tissue change during thermal ablation. Multi-modal methods such as acousto-optic (AO) and photo-acoustic (PA) imaging may provide a real-time, direct measure of lesion formation. Baseline changes in optical properties have been previously measured over limited ranges of thermal dose for tissues exposed to a temperature-controlled water bath, however, there is scant data for optical properties of lesions created by HIFU. In this work, the optical scattering and absorption coefficients from 400-1300 nm of excised chicken breast exposed to HIFU were measured using an integrating sphere spectrophotometric technique. HIFU-induced spatiotemporal temperature elevations were measured using an infrared camera and used to calculate the thermal dose delivered to a localized region of tissue. Results obtained over a range of thermal dose spanning 9 orders of magnitude show that the reduced scattering coefficient increases for HIFU exposures exceeding a threshold thermal dose of CEM43 = 600 ± 81 cumulative equivalent minutes. HIFU-induced thermal damage results in changes in scattering over all optical wavelengths, with a 2.5-fold increase for thermal lesions exceeding 70 °C. The tissue absorption coefficient was also found to increase for thermally lesioned tissue, however, the magnitude was strongly dependent on the optical wavelength and there was substantial sample-to-sample variability, such that the existence of a threshold thermal dose could not be determined. Therapeutic windows, where the optical penetration depth is expected to be greatest, were identified in the near infrared regime centered near 900 nm and 1100 nm. These data motivate further research to improve the real-time AO and PA sensing of lesion formation during HIFU therapy as an alternative to thermometry.

Modeling-based design and assessment of an acousto-optic guided high-intensity focused ultrasound system.

Real-time acousto-optic (AO) sensing has been shown to noninvasively detect changes in ex vivo tissue optical properties during high-intensity focused ultrasound (HIFU) exposures. The technique is particularly appropriate for monitoring noncavitating lesions that offer minimal acoustic contrast. A numerical model is presented for an AO-guided HIFU system with an illumination wavelength of 1064 nm and an acoustic frequency of 1.1 MHz. To confirm the model's accuracy, it is compared to previously published experimental data gathered during AO-guided HIFU in chicken breast. The model is used to determine an optimal design for an AO-guided HIFU system, to assess its robustness, and to predict its efficacy for the ablation of large volumes. It was found that a through transmission geometry results in the best performance, and an optical wavelength around 800 nm was optimal as it provided sufficient contrast with low absorption. Finally, it was shown that the strategy employed while treating large volumes with AO guidance has a major impact on the resulting necrotic volume and symmetry.

Thermal dose dependent optical property changes of ex vivo chicken breast tissues between 500 and 1100 nm.

This study examines the effectiveness of the thermal dose model in accurately predicting thermally induced optical property changes of ex vivo chicken breast between 500-1100 nm. The absorption coefficient, µa, and the reduced scattering coefficient, µ's, of samples are measured as a function of thermal dose over the range 50 °C-70 °C. Additionally, the maximum observable changes in µa and µ's are measured as a function of temperature in the range 50 °C-90 °C. Results show that the standard thermal dose model used in the majority of high-intensity focused ultrasound (HIFU) treatments is insufficient for modeling optical property changes, but that the isodose constant may be modified in order to better predict thermally induced changes. Additionally, results are presented that show a temperature dependence on changes in the two coefficients, with an apparent threshold effect occurring between 65 °C-70 °C.

Gold nanoparticle targeted photoacoustic cavitation for potential deep tissue imaging and therapy.

The laser generation of vapor bubbles around plasmonic nanoparticles can be enhanced through the application of an ultrasound field; a technique referred to as photoacoustic cavitation. The combination of light and ultrasound allows for bubble formation at lower laser fluence and peak negative ultrasound pressure than can be achieved using either modality alone. The growth and collapse of these bubbles leads to local mechanical disruption and acoustic emission, and can potentially be used to induce and monitor tissue therapy. Photoacoustic cavitation is investigated for a broad range of ultrasound pressures and nanoparticle concentrations for gold nanorods and nanospheres. The cavitation threshold fluences for both nanoparticle types are found to drastically reduce in the presence of an ultrasound field. The results indicate that photoacoustic cavitation can potentially be produced at depth in biological tissue without exceeding the safety limits for ultrasound or laser radiation at the tissue surface.

Measuring tissue properties and monitoring therapeutic responses using acousto-optic imaging.

Acousto-optic imaging is a hybrid imaging technique that exploits the interaction between light and sound to image optical contrast at depth in optically turbid media with the high spatial resolution of ultrasound. Quantitative measurement of optical properties using this technique is confounded by multiple parameters that influence the detected acousto-optic signal. In this article, we describe the origin of the acousto-optic response and review techniques that have been proposed to relate this response to the optical properties of turbid media. We present an overview of two acousto-optic sensing approaches. In the first, we demonstrate that the local transport mean free path within turbid media can be obtained by varying the pressure of the ultrasound field and processing the resulting acousto-optic signals. In the second, we demonstrate that the acousto-optic response elicited by a high-intensity ultrasound field during thermal therapy can be used to monitor the onset of lesion formation, ascertain lesion volume, and provide real-time control of exposure duration.

Modeling cavitation nucleation from laser-illuminated nanoparticles subjected to acoustic stress.

In an earlier work by Farny et al. [ARLO 6, 138-143 (2005).] it was demonstrated that the acoustic cavitation threshold in a tissue mimicking gel phantom can be lowered from 4.5 to ∼1 MPa by "seeding" the optically transparent phantom with light absorptive gold nanoparticles and irradiating these absorbers with nanosecond pulses of laser light at intensities less than 10 mJ/cm(2). As a follow-up study, a three-stage numerical model was developed to account for prenucleation heating, the nucleation and formation of the vapor cavity, and the resulting vapor bubble dynamics. Through examination of the radius-time evolution of the cavity, the combined thresholds for laser radiant exposure and acoustic peak pressure required to induce inertial cavitation are deduced. It is found that the threshold pressure decreases when laser exposure increases; but the rate depends on exposure levels and the size of the particle. Investigations of the roles of particle size and laser pulse length are performed and optimum choices for these parameters determined in order to obtain inertial cavitation at the lowest possible acoustic pressure and laser intensity.

Ambient acceleration dependence of single-bubble sonoluminescence.

Much of the research performed to study SBSL deals with the influence of external parameters (e.g., the host water temperature, the ambient pressure, the type and amount of dissolved gas in the liquid, to name a few) on the bubble dynamics and light emission. In the current paper, work carried out to study the influence of another external parameter-ambient acceleration-is described. The experiments described here were performed on the NASA KC-135 which provided both periods of reduced gravity (10(-3) g) and increased gravity (1.8 g) by flying repeated parabolic maneuvers. The resulting measurements are compared with the predictions of a numerical model and can be understood in the context of the changing hydrostatic head pressure and buoyant force acting on the bubble.

Real-time monitoring of high-intensity focused ultrasound lesion formation using acousto-optic sensing.

High-intensity focused ultrasound (HIFU) is a promising modality that is used to noninvasively ablate soft tissue tumors. Nevertheless, real-time treatment monitoring with diagnostic ultrasound still poses a significant challenge since tissue necrosis, in the absence of cavitation or boiling, provides little acoustic contrast with normal tissue. In comparison, the optical properties of tissue are significantly altered accompanying lesion formation. A photorefractive crystal-based acousto-optic (AO) sensing system that uses a single HIFU transducer to simultaneously generate tissue necrosis and pump the AO interaction is used to monitor the real-time optical changes associated with thermal lesions induced in chicken breast ex vivo. It is found that the normalized change in AO response increases proportionally with the volume of necrosis. This study demonstrates AO sensing can identify the onset and growth of lesion formation in real time and, when used as feedback to guide exposures, results in more predictable lesion formation.

Potentiating intra-arterial sonothrombolysis for acute ischemic stroke by the addition of the ultrasound contrast agents (Optison™ & SonoVue(®)).

Transcranial ultrasound in combination with intravenously administered ultrasound contrast agents (UCA) in the presence or absence of recombinant tissue plasminogen activator (rt-PA) has been widely evaluated as a new modality for treatment of ischemic stroke. Despite the successful demonstration of accelerated clot lysis there are inherent limitations associated with this modality such as inconsistency in temporal window thickness and/or potential serious cardiopulmonary reactions to intravenous administration of UCA that prevent broad application to ischemic stroke populations. As a complementary modality, we evaluated potential lysis enhancement by intra-arterial ultrasound with concurrent intra-clot delivery of UCA and rt-PA. To this end, clots were formed with average pore diameter similar to clinically retracted clots by adjusting the thrombin concentration. Physical characteristic and retention of UCA after delivery through the catheter as a function of clinically relevant flow rates of 6, 12, 18 ml/h were determined using a microscopic method. The ability of the UCA employed in this study, Optison and SonoVue, to penetrate into the clot was verified using ultrasound B-mode imaging. Clot lysis as a function of rt-PA concentration, 0.009 through 0.5 mg/ml, in the presence and absence of UCA diluted to 1:10, 1:100, and 1:200 v/v at two Peak rarefaction acoustic pressures of 1.3 and 2.1 MPa were evaluated using a weighing method. The study results suggest the addition of only 0.02 ml of 1:100 diluted UCA to rt-PA of 0.009, 0.05, 0.3, and 0.5 mg/ml can enhance the lysis rate by 3.9, 2.6, 1.9 and 1.8 fold in the presence of peak rarefaction acoustic pressure of 1.3 MPa and by 5.1, 3.4, 2.6, 3.1 in the presence of peak rarefaction acoustic pressure of 2.1 MPa, respectively. In addition, Optison and SonoVue demonstrated comparable effectiveness in enhancing the clot lysis rate. Addition of UCA to intra-arterial sonothrombolysis could be considered as a viable treatment option for ischemic stroke patients.

Ultrasonic enhancement of photoacoustic emissions by nanoparticle-targeted cavitation.

A technique to enhance the photoacoustic emissions from laser-heated nanoparticles is presented. Gold nanoparticle-doped phantoms are subjected to pulsed optical and ultrasound fields, resulting in bubble formation and collapse and producing strong acoustic emissions. The applied ultrasound field allows for cavitation at lower laser fluences than using light alone. The acoustic emission associated with bubble collapse well exceeds the direct photoacoustic response and is used to image a nanoparticle-doped region in a tissue phantom. The strong acoustic emission and low-threshold fluence associated with ultrasound-assisted cavitation make the technique well suited for nanoparticle-targeted biological imaging and tissue therapy.

Detecting cavitation in mercury exposed to a high-energy pulsed proton beam.

The Oak Ridge National Laboratory Spallation Neutron Source employs a high-energy pulsed proton beam incident on a mercury target to generate short bursts of neutrons. Absorption of the proton beam produces rapid heating of the mercury, resulting in the formation of acoustic shock waves and the nucleation of cavitation bubbles. The subsequent collapse of these cavitation bubbles promote erosion of the steel target walls. Preliminary measurements using two passive cavitation detectors (megahertz-frequency focused and unfocused piezoelectric transducers) installed in a mercury test target to monitor cavitation generated by proton beams with charges ranging from 0.041 to 4.1 muC will be reported on. Cavitation was initially detected for a beam charge of 0.082 muC by the presence of an acoustic emission approximately 250 mus after arrival of the incident proton beam. This emission was consistent with an inertial cavitation collapse of a bubble with an estimated maximum bubble radius of 0.19 mm, based on collapse time. The peak pressure in the mercury for the initiation of cavitation was predicted to be 0.6 MPa. For a beam charge of 0.41 muC and higher, the lifetimes of the bubbles exceeded the reverberation time of the chamber ( approximately 300 mus), and distinct windows of cavitation activity were detected, a phenomenon that likely resulted from the interaction of the reverberation in the chamber and the cavitation bubbles.

The correlation between bubble-enhanced HIFU heating and cavitation power.

It has been established that while the inherent presence of bubbles increases heat generation due to scattering and absorption, inertial cavitation is responsible for elevated heating during high-intensity focused ultrasound (HIFU) application. The contribution of bubble-induced heating can be an important factor to consider, as it can be several times greater than the expected heat deposition from absorption of energy from the primary ultrasound field. The temperature and cavitation signal near the focus were measured for 5.5-s continuous-wave 1.1-MHz HIFU sonications in tissue mimicking phantoms. The measured temperature was corrected for heating predicted from the primary ultrasound absorption to isolate the temperature rise from the bubble activity. The temperature rise induced from cavitation correlates well with a measurement of the instantaneous "cavitation power" as indicated by the mean square voltage output of a 15-MHz passive cavitation detector. The results suggest that careful processing of the cavitation signals can serve as a proxy for measuring the heating contribution from inertial cavitation.

Quantitative characterization of turbid media using pressure contrast acousto-optic imaging.

An acousto-optic imaging technique suitable for the local and quantitative determination of subsurface optical properties in turbid media is presented. Acousto-optic signals elicited by ultrasound pulses at two different peak pressures in turbid media are detected by using photorefractive-crystal-based interferometry. The ratio of the measured signals, once calibrated for a particular set of pressure pulses, is found to give a direct measure of the reduced scattering coefficient of the interaction region between the light and sound. Measurements of the reduced scattering coefficient of inclusions buried in diffuse tissue phantoms are demonstrated.

Sensing a buried resonant object by single-channel time reversal.

Scaled laboratory experiments are conducted to assess the efficacy of iterative, single-channel time reversal for enhancement of monostatic returns from resonant spheres in the free field and buried in a sediment phantom. Experiments are performed in a water tank using a broad-band piston transducer operating between 0.4 and 1.5 MHz and calibrated using free surface reflections. Solid and hollow metallic spheres, 6.35 mm in diameter, are buried in a consolidation of 128-microm-mean- diameter spherical glass beads. The procedure consists of exciting the target object with a broadband pulse, sampling the return using a finite time window, reversing the signal in time, and using this reversed signal as the source waveform for the next interrogation. Results indicate that the spectrum of the returns rapidly converges to the dominant mode in the backscattering response of the target. Signal-to-noise enhancement of the target echo is demonstrated for a target at several burial depths. Images generated by scanning the transducer over the location of multiple buried targets demonstrate the ability of the technique to distinguish between targets of differing type and to yield an enhancement of different modes within the response of a single target as a function of transducer position and processing bandwidth.

Temporal and spatial detection of HIFU-induced inertial and hot-vapor cavitation with a diagnostic ultrasound system.

The onset and presence of inertial cavitation and near-boiling temperatures in high-intensity focused ultrasound (HIFU) therapy have been identified as important indicators of energy deposition for therapy guidance. Passive cavitation detection is commonly used to detect bubble emissions, where a fixed-focus single-element acoustic transducer is typically used as a passive cavitation detector (PCD). This technique is suboptimal for clinical applications, because most PCD transducers are tightly focused and afford limited spatial coverage of the HIFU focal region. A Terason 2000 Ultrasound System was used as a PCD array to expand the spatial detection region for cavitation by operating in passive mode, obtaining the radiofrequency signals corresponding to each scan line and filtering the contribution from scattering of the HIFU signal harmonics. This approach allows for spatially resolved detection of both inertial and stable cavitation throughout the focal region. Measurements with the PCD array during sonication with a 1.1-MHz HIFU source in tissue phantoms were compared with single-element PCD and thermocouple sensing. Stable cavitation signals at the harmonics and superharmonics increased in a threshold fashion for temperatures >90 degrees C, an effect attributed to high vapor pressure in the cavities. Incorporation of these detection techniques in a diagnostic ultrasound platform could result in a powerful tool for improving HIFU guidance and treatment.