Spectral Ultrasound Characterization of Tissues and Tissue Engineered Constructs.

Even though ultrasound imaging is widely used in clinical diagnosis and image-guided interventions, the field is far behind other areas of clinical quantitative image analysis, such as MRI, CT and X-ray mammography. In this thesis, non-destructive and non-invasive ultrasound characterization techniques were developed to study the tissue micro-structural details using high frequency spectral ultrasound imaging (SUSI). The techniques were explored in in-vitro conditions of acellular and cellular tissue engineered constructs and then on ex-vivo tissues for their characterization. SUSI was used to assess the amount of hydroxyl-apatite (HA) mineral, differentiate HA mineral types and study their distribution in acellular tissue engineered constructs. The process of mineral deposition from surrounding mineralizing media onto simple collagen constructs was also studied and characterized with SUSI. 3D morphological changes of the constructs with MC3t3 cells was monitored and characterized for the developmental changes such as net cell proliferation/apoptosis and cell differentiation process through mineral production by the early osteoblastic MC3t3-cell constructs in-situ. A novel method was introduced using SUSI to estimate the amount of mineral secreted by the differentiated osteoblast cells in a non-destructive method. Then, SUSI was investigated in ex-vivo cardiac tissues to monitor and characterize the cellular changes during high-intensity focused ultrasound ablation with high-frame-rate and high-resolution ultrasound imaging. The mechanistic hypotheses behind the improvement in lesion detection were investigated and best identification methods to assess lesion formation and transient gas body activities were proposed to provide a method for visualizing spatiotemporal evolution of lesion and gas–body activity and for predicting macroscopic cavity formation upon its implementation as a real-time monitoring technique with feedback control system for HIFU treatment of atrial fibrillation to improve the ablation process. Even though the results from the developed techniques show great promise in in-vitro and ex-vivo settings, additional work needs to be carried out to demonstrate the applicability of the techniques in in-vivo.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/99788/1/msreddy_1.pd

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). Radiofrequency (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%, as compared to macroscopic images of lesions). A criterion based on a line-to-line decorrelation coefficient is proposed for identification of transient gas bodies

High-Frequency Rapid B-Mode Ultrasound Imaging for Real-Time Monitoring of Lesion Formation and Gas Body Activity During High-Intensity Focused Ultrasound Ablation

Abstract: 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/cm2). RF data from Bmode 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 (millimetersized) 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

High-frequency ultrasound M-mode monitoring of HIFU ablation in cardiac tissue

Effective real-time HIFU lesion detection is important for expanded use of HIFU in interventional electrophysiology (e.g., epicardial ablation of cardiac arrhythmia). The goal of this study was to investigate rapid, high-frequency M-mode ultrasound imaging for monitoring spatiotemporal changes in tissue during HIFU application. The HIFU application (4.33 MHz, 1000 Hz PRF, 50% duty cycle, 1 s exposure, 6100 W/cm2) was perpendicularly applied to porcine cardiac tissue with a high-frequency imaging system (Visualsonics Vevo 770, 55 MHz, 4.5 mm focal distance) confocally aligned. Radiofrequency (RF) M-mode data (1 kHz PRF, 4 s × 7 mm) was acquired before, during, and after HIFU treatment. Gross lesions were compared with M-mode data to correlate lesion and cavity formation. Integrated backscatter, echo-decorrelation parameters, and their cumulative extrema over time were analyzed for automatically identifying lesion width and bubble formation. Cumulative maximum integrated backscatter showed the best results for identifying the final lesion width, and a criterion based on line-to-line decorrelation was proposed for identification of transient bubble activity

Lymph Node Characterization in Vivo Using Endoscopic Ultrasound Spectrum Analysis With Electronic Array Echo Endoscopes

Our purpose was to demonstrate the use of radiofrequency spectral analysis to distinguish between benign and malignant lymph nodes with data obtained using electronic array echo endoscopes, as we have done previously using mechanical echo endoscopes. In a prospective study, images were obtained from eight patients with benign-appearing lymph nodes and 11 with malignant lymph nodes, as verified by fine-needle aspiration. Midband fit, slope, intercept, correlation coefficient, and root-mean-square (RMS) deviation from a linear regression of the calibrated power spectra were determined and compared between the groups. Significant differences were observable for mean midband fit, intercept, and RMS deviation (t test P \u3c 0.05). For benign (n = 16) vs. malignant (n = 12) lymph nodes, midband fit and RMS deviation provided classification with 89 % accuracy and area under receiver operating characteristic (ROC) curve of 0.95 based on linear discriminant analysis. We concluded that the mean spectral parameters of the backscattered signals from electronic array echo endoscopy can provide a noninvasive method to quantitatively discriminate between benign and malignant lymph nodes

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

Comparison of cell viability at day 1 and 21 of MC3T3 cells seeded in collagen constructs, in either control or osteogenic media (cytoplasm of living cells is stained green, nuclei of dead cells is stained red).

<p>Constructs in (A) were not imaged using ultrasound, while those in (B) were imaged using ultrasound. Bar plot in (C) shows quantification of cell viability calculated from the images. Scale bar  = 200 µm. Best viewed in color.</p

Developmental changes in sizes of MC3T3 cells seeded in collagen constructs.

<p>(A) –(D) Fluorescence staining of MC3T3 cells embedded in collagen constructs in control and osteogenic media on day 1 and day 21 (actin cytoskeleton is stained green, nuclei are stained blue). (E) Estimated diameter of cells from SUSI analysis over time in culture. Scale bar  = 50 µm. Best viewed in color.</p

Noninvasive Quantification of <i>In Vitro</i> Osteoblastic Differentiation in 3D Engineered Tissue Constructs Using Spectral Ultrasound Imaging

<div><p>Non-destructive monitoring of engineered tissues is needed for translation of these products from the lab to the clinic. In this study, non-invasive, high resolution spectral ultrasound imaging (SUSI) was used to monitor the differentiation of MC3T3 pre-osteoblasts seeded within collagen hydrogels. SUSI was used to measure the diameter, concentration and acoustic attenuation of scatterers within such constructs cultured in either control or osteogenic medium over 21 days. Conventional biochemical assays were used on parallel samples to determine DNA content and calcium deposition. Construct volume and morphology were accurately imaged using ultrasound. Cell diameter was estimated to be approximately 12.5–15.5 µm using SUSI, which corresponded well to measurements of fluorescently stained cells. The total number of cells per construct assessed by quantitation of DNA content decreased from 5.6±2.4×10⁴ at day 1 to 0.9±0.2×10⁴ at day 21. SUSI estimation of the equivalent number of acoustic scatters showed a similar decreasing trend, except at day 21 in the osteogenic samples, which showed a marked increase in both scatterer number and acoustic impedance, suggestive of mineral deposition by the differentiating MC3T3 cells. Estimation of calcium content by SUSI was 41.7±11.4 µg/ml, which agreed well with the biochemical measurement of 38.7±16.7 µg/ml. Color coded maps of parameter values were overlaid on B-mode images to show spatiotemporal changes in cell diameter and calcium deposition. This study demonstrates the use of non-destructive ultrasound imaging to provide quantitative information on the number and differentiated state of cells embedded within 3D engineered constructs, and therefore presents a valuable tool for longitudinal monitoring of engineered tissue development.</p></div