Ronald A. Roy

Summary: 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 cm ³ and 300 cm ³ , 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.

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.

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.

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%.

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.

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.

Real-time acousto-optic (AO) sensing has been shown to non-invasively detect changes in ex vivo tissue optical properties during high-intensity focused ultrasound (HIFU) exposures. Baseline changes in optical properties have been previously measured as a function of thermal-dose for tissues exposed to a temperature-controlled water bath (doi:10.1088/0031-9155/59/13/3249). In this work, the wavelength-dependent optical scattering and absorption of ex vivo tissues exposed to HIFU were measured using an integrating sphere spectrophotometric technique employed previously. 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. We consider the impact of thermal dose, temperature elevation, and heating rate on the formation of HIFU lesions and the resulting changes in tissue optical properties. Recently, reported results (doi:10.1088/0031-9155/59/13/3249) refute the hypothesis that optical property changes in tissue are based solely on accumulated thermal dose—this claim will be explored further through the optical characterization of lesions formed in clinically relevant tissues. Results will show how wavelength-dependent optical property changes in tissues can be used to improve the AO sensing of lesion formation during HIFU therapy as an alternative to thermometry. [Work supported by the ASA F.V. Hunt Fellowship and the University of Oxford.]

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.