Teaching an advanced undergraduate acoustics laboratory without a laboratory: Course developments enabling teaching during the COVID-19 pandemic.

This paper describes ongoing developments to an advanced laboratory course at Kettering University, which is targeted to students in engineering and engineering physics and emphasizes theoretical, computational, and experimental components in the context of airborne acoustics and modal testing [cf. D. A. Russell and D. O. Ludwigsen, J. Acoust. Soc. Am. 131, 2515-2524 (2012)]. These developments have included a transition to electronic laboratory notebooks and cloud-based computing resources, incorporation of updated hardware and software, and creation and testing of a multiple-choice assessment instrument for the course. When Kettering University suddenly shifted to exclusively remote teaching in March 2020 due to the COVID-19 pandemic, many of these changes proved to be essential for enabling rapid adaptation to a situation in which a laboratory was not available for the course. Laboratory activities were rewritten by crowdsourcing archived data, videos were incorporated to illustrate dynamic phenomena, and computer simulations were used to retain student interactivity. The comparison of multiple measures, including the assessment instrument, team-based grades on project papers, and individual grades on final exams, indicates that most students were successful at learning the course material and adapting to work on team-based projects in the midst of challenging remote learning conditions.

Magnetic Hyperthermia in Y79 Retinoblastoma and ARPE-19 Retinal Epithelial Cells: Tumor Selective Apoptotic Activity of Iron Oxide Nanoparticle.

PURPOSE: To evaluate selective apoptosis of Y79 retinoblastoma versus ARPE-19 retinal pigment epithelial cells by using different doses of dextran-coated iron oxide nanoparticles (DCIONs) in a magnetic hyperthermia paradigm. METHODS: Y79 and ARPE-19 cells were exposed to different concentrations of DCIONs, namely, 0.25, 0.5, 0.75, and 1 mg/ml. After 2 hours of incubation, cells were exposed to a magnetic field with a frequency of 250 kHz and an amplitude of 4 kA/m for 30 minutes to raise the cellular temperature between 42 and 46°C. Y79 and ARPE-19 cells incubated with DCION without magnetic field exposure were used as controls. Cell viability and apoptosis were assessed at 4, 24, and 72 hours after hyperthermia treatment. RESULTS: At 4 hours following magnetic hyperthermia, cell death for Y79 cells was 1%, 8%, 17%, and 17% for 0.25, 0.5, 0.75 and 1 mg/ml of DCION, respectively. Cell death increased to 47%, 59%, 70%, and 75% at 24 hours and 16%, 45%, 50%, and 56% at 72 hours for 0.25, 0.5, 0.75, and 1 mg/ml of DCIONs, respectively. Magnetic hyperthermia did not have any significant toxic effects on ARPE-19 cells at all DCION concentrations, and minimal baseline cytotoxicity of DCIONs on Y79 and ARPE-19 cells was observed without magnetic field activation. Gene expression profiling showed that genes involved in FAS and tumor necrosis factor alpha signaling pathways were activated in Y79 cells following hyperthermia. Caspase 3/7 activity in Y79 cells increased following treatment, consistent with the activation of caspase-mediated apoptosis and loss of cell viability by magnetic hyperthermia. CONCLUSION: Magnetic hyperthermia using DCIONs selectively kills Y79 cells at 0.5 mg/ml or higher concentrations via the activation of apoptotic pathways. TRANSLATIONAL RELEVANCE: Magnetic hyperthermia using DCIONs might play a role in targeted management of retinoblastoma.

Electrophysiological changes correlated with temperature increases induced by high-intensity focused ultrasound ablation.

To gain better understanding of the detailed mechanisms of high-intensity focused ultrasound (HIFU) ablation for cardiac arrhythmias, we investigated how the cellular electrophysiological (EP) changes were correlated with temperature increases and thermal dose (cumulative equivalent minutes [CEM43]) during HIFU application using Langendorff-perfused rabbit hearts. Employing voltage-sensitive dye di-4-ANEPPS, we measured the EP and temperature during HIFU using simultaneous optical mapping and infrared imaging. Both action potential amplitude (APA) and action potential duration at 50% repolarization (APD50) decreased with temperature increases, and APD50 was more thermally sensitive than APA. EP and tissue changes were irreversible when HIFU-induced temperature increased above 52.3 ± 1.4°C and log10(CEM43) above 2.16 ± 0.51 (n = 5), but were reversible when temperature was below 50.1 ± 0.8°C and log10(CEM43) below -0.9 ± 0.3 (n = 9). EP and temperature/thermal dose changes were spatially correlated with HIFU-induced tissue necrosis surrounded by a transition zone.

Tomographic reconstruction of tissue properties and temperature increase for high-intensity focused ultrasound applications.

The acoustic and thermal properties as well as the temperature change within a tissue volume during high-intensity focused ultrasound ablation are critically important for treatment planning and monitoring. Described in this article is a tomographic reconstruction method used to determine the tissue properties and increase in temperature in a 3-D volume. On the basis of the iterative finite-element solution to the bioheat equation coupled with Tikhonov regularization techniques, our reconstruction algorithm solves the inverse problem of bioheat transfer and uses the time-dependent temperature measured on a tissue surface to obtain the acoustic absorption coefficient, thermal diffusivity and temperature increase within the subsurface volume. Numerical simulations were performed to validate the reconstruction algorithm. The method was initially conducted in ex vivo experiments in which time-dependent temperature on a tissue surface was measured using high-resolution, non-invasive infrared thermography.

Characterization of Lesion Formation and Bubble Activities during High Intensity Focused Ultrasound Ablation using Temperature-Derived Parameters.

Successful high-intensity focused ultrasound (HIFU) thermal tissue ablation relies on accurate information of the tissue temperature and tissue status. Often temperature measurements are used to predict and monitor the ablation process. In this study, we conducted HIFU ablation experiments with ex vivo porcine myocardium tissue specimens to identify changes in temperature associated with tissue coagulation and bubble/cavity formation. Using infrared (IR) thermography and synchronized bright-field imaging with HIFU applied near the tissue surface, parameters derived from the spatiotemporal evolution of temperature were correlated with HIFU-induced lesion formation and overheating, of which the latter typically results in cavity generation and/or tissue dehydration. Emissivity of porcine myocardium was first measured to be 0.857 ± 0.006 (n = 3). HIFU outcomes were classified into non-ablative, normal lesion, and overheated lesion. A marked increase in the rate of temperature change during HIFU application was observed with lesion formation. A criterion using the maximum normalized second time derivative of temperature change provided 99.1% accuracy for lesion identification with a 0.05 s-1 threshold. Asymmetric temperature distribution on the tissue surface was observed to correlate with overheating and/or bubble generation. A criterion using the maximum displacement of the spatial location of the peak temperature provided 90.9% accuracy to identify overheated lesion with a 0.16 mm threshold. Spatiotemporal evolution of temperature obtained using IR imaging allowed determination of the cumulative equivalent minutes at 43 °C (CEM43) for lesion formation to be 170 min. Similar temperature characteristics indicative of lesion formation and overheating were identified for subsurface HIFU ablation. These results suggest that parameters derived from temperature changes during HIFU application are associated with irreversible changes in tissue and may provide useful information for monitoring HIFU treatment.

High-frequency rapid B-mode ultrasound imaging for real-time monitoring of lesion formation and gas body activity during high-intensity focused ultrasound ablation.

The goal of this study was to examine the ability of high-frame-rate, high-resolution imaging to monitor tissue necrosis and gas-body activities formed during high-intensity focused ultrasound (HIFU) application. Ex vivo porcine cardiac tissue specimens (n = 24) were treated with HIFU exposure (4.33 MHz, 77 to 130 Hz pulse repetition frequency (PRF), 25 to 50% duty cycle, 0.2 to 1 s, 2600 W/cm(2)). RF data from B-mode ultrasound imaging were obtained before, during, and after HIFU exposure at a frame rate ranging from 77 to 130 Hz using an ultrasound imaging system with a center frequency of 55 MHz. The time history of changes in the integrated backscatter (IBS), calibrated spectral parameters, and echo-decorrelation parameters of the RF data were assessed for lesion identification by comparison against gross sections. Temporal maximum IBS with +12 dB threshold achieved the best identification with a receiver-operating characteristic (ROC) curve area of 0.96. Frame-to-frame echo decorrelation identified and tracked transient gas-body activities. Macroscopic (millimeter-sized) cavities formed when the estimated initial expansion rate of gas bodies (rate of expansion in lateral-to-beam direction) crossed 0.8 mm/s. Together, these assessments provide a method for monitoring spatiotemporal evolution of lesion and gas-body activity and for predicting macroscopic cavity formation.

Characterization of the pancreas in vivo using EUS spectrum analysis with electronic array echoendoscopes.

BACKGROUND: Spectral analysis of the radiofrequency (RF) signals that underlie grayscale EUS images has been used to provide quantitative, objective information about tissue histology. OBJECTIVE: Our purpose was to validate RF spectral analysis as a method to distinguish between chronic pancreatitis (CP) and pancreatic cancer (PC). DESIGN AND SETTING: A prospective study of eligible patients was conducted to analyze the RF data obtained by using electronic array echoendoscopes. PATIENTS: Pancreatic images were obtained by using electronic array echoendoscopes from 41 patients in a prospective study, including 15 patients with PC, 15 with CP, and 11 with a normal pancreas. MAIN OUTCOME MEASUREMENTS: Midband fit, slope, intercept, correlation coefficient, and root mean square deviation from a linear regression of the calibrated power spectra were determined and compared among the groups. RESULTS: Statistical analysis showed that significant differences were observable between groups for mean midband fit, intercept, and root mean square deviation (t test, P < .05). Discriminant analysis of these parameters was then performed to classify the data. For CP (n = 15) versus PC (n = 15), the same parameters provided 83% accuracy and an area under the curve of 0.83. LIMITATIONS: Moderate sample size and spatial averaging inherent in the technique. CONCLUSIONS: This study shows that mean spectral parameters of the backscattered signals obtained by using electronic array echoendoscopes can provide a noninvasive method to quantitatively discriminate between CP and PC.

High-frequency ultrasound m-mode imaging for identifying lesion and bubble activity during high-intensity focused ultrasound ablation.

Effective real-time monitoring of high-intensity focused ultrasound (HIFU) ablation is important for application of HIFU technology in interventional electrophysiology. This study investigated rapid, high-frequency M-mode ultrasound imaging for monitoring spatiotemporal changes during HIFU application. HIFU (4.33 MHz, 1 kHz PRF, 50% duty cycle, 1 s, 2600‒6100 W/cm²) was applied to ex vivo porcine cardiac tissue specimens with a confocally and perpendicularly aligned high-frequency imaging system (Visualsonics Vevo 770, 55 MHz center frequency). Radio-frequency (RF) data from M-mode imaging (1 kHz PRF, 2 s × 7 mm) was acquired before, during and after HIFU treatment (n = 12). Among several strategies, the temporal maximum integrated backscatter with a threshold of +12 dB change showed the best results for identifying final lesion width (receiver-operating characteristic curve area 0.91 ± 0.04, accuracy 85 ± 8%, compared with macroscopic images of lesions). A criterion based on a line-to-line decorrelation coefficient is proposed for identification of transient gas bodies.

Frequency-domain analysis of photoacoustic imaging data from prostate adenocarcinoma tumors in a murine model.

Photoacoustic imaging is an emerging technique for anatomical and functional sub-surface imaging but previous studies have predominantly focused on time-domain analysis. In this study, frequency-domain analysis of the radio-frequency signals from photoacoustic imaging was performed to generate quantitative parameters for tissue characterization. To account for the response of the imaging system, the photoacoustic spectra were calibrated by dividing the photoacoustic spectra (radio-frequency ultrasound spectra resulting from laser excitation) from tissue by the photoacoustic spectrum of a point absorber excited under the same conditions. The resulting quasi-linear photoacoustic spectra were fit by linear regression and midband fit, slope and intercept were computed from the best-fit line. These photoacoustic spectral parameters were compared between the region-of-interests (ROIs) representing prostate adenocarcinoma tumors and adjacent normal flank tissue in a murine model. The mean midband fit and intercept in the ROIs showed significant differences between cancerous and noncancerous regions. These initial results suggest that such frequency-domain analysis can provide a quantitative method for tumor tissue characterization using photoacoustic imaging in vivo.

Modulation of intracellular Ca2+ concentration in brain microvascular endothelial cells in vitro by acoustic cavitation.

Localized delivery of therapeutic agents through the blood-brain barrier (BBB) is a clinically significant task that remains challenging. Ultrasound (US) application after intravenous administration of microbubbles has been shown to generate localized BBB opening in animal models but the detailed mechanisms are not yet fully described. The current study investigates the effects of US-stimulated microbubbles on in vitro murine brain microvascular endothelial (bEnd.3) cells by monitoring sonoporation and changes in intracellular calcium concentration ([Ca(2+)](i)) using real-time fluorescence and high-speed brightfield microscopy. Cells seeded in microchannels were exposed to a single US pulse (1.25 MHz, 10 cycles, 0.24 MPa peak negative pressure) in the presence of Definity microbubbles and extracellular calcium concentration [Ca(2+)](o) = 0.9 mM. Disruption of the cell membrane was assessed using propidium iodide (PI) and change in the [Ca(2+)](i) was measured using fura-2. Cells adjacent to a microbubble exhibited immediate [Ca(2+)](i) changes after US pulse with and without PI uptake and the [Ca(2+)](i) changes were twice as large in cells with PI uptake. Cell viability assays showed that sonoporated cells could survive with modulation of [Ca(2+)](i) and uptake of PI. Cells located near sonoporated cells were observed to exhibit changes in [Ca(2+)](i) that were delayed from the time of US application and without PI uptake. These results demonstrate that US-stimulated microbubbles not only directly cause changes in [Ca(2+)](i) in brain endothelial cells in addition to sonoporation but also generate [Ca(2+)](i) transients in cells not directly interacting with microbubbles, thereby affecting cells in larger regions beyond the cells in contact with microbubbles.

In vivo characterization of pancreatic and lymph node tissue by using EUS spectrum analysis: a validation study.

BACKGROUND: Quantitative spectral analysis of the radiofrequency (RF) signals that underlie grayscale EUS images can be used to provide additional, objective information about tissue state. OBJECTIVE: Our purpose was to validate RF spectral analysis as a method to distinguish between (1) benign and malignant lymph nodes and (2) normal pancreas, chronic pancreatitis, and pancreatic cancer. DESIGN AND SETTING: A prospective validation study of eligible patients was conducted to compare with pilot study RF data. PATIENTS: Forty-three patients underwent EUS of the esophagus, stomach, pancreas, and surrounding intra-abdominal and mediastinal lymph nodes (19 from a previous pilot study and 24 additional patients). MAIN OUTCOME MEASUREMENTS: Midband fit, slope, intercept, and correlation coefficient from a linear regression of the calibrated RF power spectra were determined. RESULTS: Discriminant analysis of mean pilot-study parameters was then performed to classify validation-study parameters. For benign versus malignant lymph nodes, midband fit and intercept (both with t test P < .058) provided classification with 67% accuracy and area under the receiver operating curve (AUC) of 0.86. For diseased versus normal pancreas, midband fit and correlation coefficient (both with analysis of variance P < .001) provided 93% accuracy and an AUC of 0.98. For pancreatic cancer versus chronic pancreatitis, the same parameters provided 77% accuracy and an AUC of 0.89. Results improved further when classification was performed with all data. LIMITATIONS: Moderate sample size and spatial averaging inherent to the technique. CONCLUSIONS: This study confirms that mean spectral parameters provide a noninvasive method to quantitatively discriminate benign and malignant lymph nodes as well as normal and diseased pancreas.

Characterization of pancreatic cancer and intra-abdominal lymph node malignancy using spectrum analysis of endoscopic ultrasound imaging.

This study assessed the ability of spectral analysis of endoscopic ultrasound (EUS) RF signals acquired in humans in vivo to distinguish between (1) benign and malignant intraabdominal and mediastinal lymph nodes and (2) pancreatic cancer, chronic pancreatitis, and normal pancreas. Mean midband fit, slope, intercept, and correlation coefficient from a linear regression of the calibrated RF power spectra were computed over regions of interest defined by the endoscopist. Linear discriminant analysis was then performed to develop a classification of the resulting spectral parameters. For lymph nodes, classification based on the midband fit and intercept provided 67% sensitivity, 82% specificity, and 73% accuracy for malignant vs. benign nodes. For pancreas, classification based on midband fit and correlation coefficient provided 95% sensitivity, 93% specificity, and 93% accuracy for diseased vs. normal pancreas and 85% sensitivity, 71% specificity, and 85% accuracy for pancreatic cancer vs. chronic pancreatitis. These promising results suggest that mean spectral parameters can provide a non-invasive method to quantitatively characterize pancreatic cancer and lymph malignancy in vivo.

The size of sonoporation pores on the cell membrane.

Sonoporation uses ultrasound (US) to generate transient nonselective pores on the cell membrane and has been exploited as a nonviral intracellular drug and gene delivery strategy. The pore size determines the size of agents that can be delivered into the cytoplasm using the technique. However, measurements of the dynamic, submicron-scale pores have not been readily available. Electron microscopy or atomic force microscopy has been used to gauge pore size but such techniques are intrinsically limited to post-US measurements that may not accurately reveal the relevant information. As previously demonstrated, changes of the transmembrane current (TMC) of a single cell under voltage clamp can be used for monitoring sonoporation in real-time. Because the TMC is related to the diffusion of ions through the pores on the membrane, it can potentially provide information of the pore size generated in sonoporation. Using Xenopus laevis oocytes as the model system, the TMC of single cells under voltage clamp was measured in real-time to assess formation of pores on the membrane in sonoporation. The cells were exposed to US (0.2 s, 0.3 MPa, 1.075 MHz) in the presence of Definity microbubbles. Experiments were designed to obtain the TMC corresponding to a single pore on the membrane. The size of the pores was estimated from an electro-diffusion model that relates the TMC with pore size from the ion transport through the pores on the membrane. The mean radius of single pores was determined to be 110 nm with standard deviation of 40 nm. This study reports the first results of pore size from the TMC measured using the voltage clamp technique.

EUS spectrum analysis for in vivo characterization of pancreatic and lymph node tissue: a pilot study.

BACKGROUND: EUS is limited by variability in the examiner's subjective interpretation of B-scan images to differentiate among normal, inflammatory, and malignant tissue. By using information otherwise discarded by conventional EUS systems, quantitative spectral analysis of the raw radiofrequency (RF) signals underlying EUS images enables tissue to be characterized more objectively. OBJECTIVE: Our purpose was to determine the feasibility of using spectral analysis of EUS data for characterization of pancreatic tissue and lymph nodes. DESIGN AND SETTING: A pilot study of eligible patients was conducted to analyze the RF data obtained during EUS by using spectral parameters. PATIENTS: Twenty-one subjects who underwent EUS of the esophagus, stomach, pancreas, and surrounding intra-abdominal and mediastinal lymph nodes. MAIN OUTCOME MEASUREMENTS: Linear regression parameters of calibrated power spectra of the RF signals were tested to differentiate normal pancreas from chronic pancreatitis and from pancreatic cancer as well as benign from malignant-appearing lymph nodes. RESULTS: The mean intercept, slope, and midband fit of the spectra differed significantly among normal pancreas, adenocarcinoma, and chronic pancreatitis when all were compared with each other (P < .01). On direct comparison, mean midband fit for adenocarcinoma differed significantly from that for chronic pancreatitis (P < .05). For lymph nodes, mean midband fit and intercept differed significantly between benign- and malignant-appearing lymph nodes (P < .01 and P < .05, respectively). LIMITATIONS: Small sample population and spatial averaging inherent to this technique. CONCLUSIONS: Mean spectral parameters in EUS imaging can provide a noninvasive method to discriminate normal from diseased pancreas and lymph nodes.