Mean Scatterer Spacing Estimation Using Cepstrum-Based Continuous Wavelet Transform.

The goal of this study was to develop an ultrasound (US) scatterer spacing estimation method using an enhanced cepstral analysis based on continuous wavelet transforms (CWTs). Simulations of backscattering media containing periodic and quasi-periodic scatterers were carried out to test the developed algorithm. Experimental data from HT-29 pellets and in vivo PC3 tumors were then used to estimate the mean scatterer spacing. For simulated media containing quasi-periodic scatterers at 1-mm and 100- [Formula: see text] spacing with 5% positional variation, the developed algorithm yielded a spacing estimation error of ~1% for 25- and 55-MHz US pulses. The mean scatterer spacing of HT-29 cell pellets (31.97 [Formula: see text]) was within 3% of the spacing obtained from histology and agreed with the predicted spacing from simulations based on the same pellets for both frequencies. The agreement extended to in vivo PC3 tumors estimation of the spacing with a variance of 1.68% between the spacing derived from the tumor histology and the application of the CWT to the experimental results. The developed technique outperformed the traditional cepstral methods as it can detect nonprominent peaks from quasi-random scatterer configurations. This work can be potentially used to detect morphological tissue changes during normal development or disease treatment.

High frequency ultrasound imaging and simulations of sea urchin oocytes.

High frequency ultrasound backscatter signals from sea urchin oocytes were measured using a 40 MHz transducer and compared to numerical simulations. The Faran scattering model was used to calculate the ultrasound scattered from single oocytes in suspension. The urchin oocytes are non-nucleated with uniform size and biomechanical properties; the backscatter from each cell is similar and easy to simulate, unlike typical nucleated mammalian cells. The time domain signal measured from single oocytes in suspension showed two distinct peaks, and the power spectrum was periodic with minima spaced approximately 10 MHz apart. Good agreement to the Faran scattering model was observed. Measurements from tightly packed oocyte cell pellets showed similar periodic features in the power spectra, which was a result of the uniform size and consistent biomechanical properties of the cells. Numerical simulations that calculated the ultrasound scattered from individual oocytes within a three dimensional volume showed good agreement to the measured signals and B-scan images. A cepstral analysis of the signal was used to calculate the size of the cells, which was 78.7 µm (measured) and 81.4 µm (simulated). This work supports the single scattering approximation, where ultrasound is discretely scattered from single cells within a bulk homogeneous sample, and that multiple scattering has a negligible effect. This technique can be applied towards understanding the complex scattering behaviour from heterogeneous tissues.

Monitoring Quantitative Ultrasound Parameter Changes in a Cell Pellet Model of Cell Starvation.

Although it has previously been shown that the spectral analysis of ultrasound backscatter data is sensitive to the cellular changes caused by apoptosis, the sensitivity of spectral analysis to oncosis or ischemic cell death had not previously been studied. Whereas many anticancer treatments induce apoptosis, others induce cell starvation, or oncosis. HT-29 colon adenocarcinoma cells were formed into pellets and covered in phosphate-buffered saline at room temperature for 56 h. The pellets were imaged every 8 h with high-frequency (55 MHz) ultrasound and the raw radio-frequency data processed. The lack of nutrients available to the cells induced cell death due to oncosis. The attenuation slope, speed of sound, spectral slope, and midband fit were estimated at each of the eight time points to identify changes as the cells died due to starvation. The spectral slope decreased monotonically over the 56 h, whereas the attenuation slope showed an increase between 1 and 48 h, followed by a slight decrease between 48 and 56 h. The midband fit did not vary over time. The speed of sound increased from 1514 to 1532 m/s over the first 24 h, after which time it plateaued. These in vitro results indicate different trends in ultrasound parameter changes from those of in vitro apoptotic cells, suggesting that these different methods of cell death can be identified not only by morphological markers, but also by specific ultrasound signatures.

Photoacoustic signal characterization of cancer treatment response: Correlation with changes in tumor oxygenation.

Frequency analysis of the photoacoustic radiofrequency signals and oxygen saturation estimates were used to monitor the in-vivo response of a novel, thermosensitive liposome treatment. The liposome encapsulated doxorubicin (HaT-DOX) releasing it rapidly (<20 s) when the tumor was exposed to mild hyperthermia (43 °C). Photoacoustic imaging (VevoLAZR, 750/850 nm, 40 MHz) of EMT-6 breast cancer tumors was performed 30 min pre- and post-treatment and up to 7 days post-treatment (at 2/5/24 h timepoints). HaT-DOX-treatment responders exhibited on average a 22% drop in oxygen saturation 2 h post-treatment and a decrease (45% at 750 nm and 73% at 850 nm) in the slope of the normalized PA frequency spectra. The spectral slope parameter correlated with treatment-induced hemorrhaging which increased the optical absorber effective size via interstitial red blood cell leakage. Combining frequency analysis and oxygen saturation estimates differentiated treatment responders from non-responders/control animals by probing the treatment-induced structural changes of blood vessel.

Correction: Photoacoustic Imaging of Cancer Treatment Response: Early Detection of Therapeutic Effect from Thermosensitive Liposomes.

[This corrects the article DOI: 10.1371/journal.pone.0165345.].

Photoacoustic Imaging of Cancer Treatment Response: Early Detection of Therapeutic Effect from Thermosensitive Liposomes.

Imaging methods capable of indicating the potential for success of an individualized treatment course, during or immediately following the treatment, could improve therapeutic outcomes. Temperature Sensitive Liposomes (TSLs) provide an effective way to deliver chemotherapeutics to a localized tumoral area heated to mild-hyperthermia (HT). The high drug levels reached in the tumor vasculature lead to increased tumor regression via the cascade of events during and immediately following treatment. For a TSL carrying doxorubicin (DOX) these include the rapid and intense exposure of endothelial cells to high drug concentrations, hemorrhage, blood coagulation and vascular shutdown. In this study, ultrasound-guided photoacoustic imaging was used to probe the changes to tumors following treatment with the TSL, HaT-DOX (Heat activated cytoToxic). Levels of oxygen saturation (sO2) were studied in a longitudinal manner, from 30 min pre-treatment to 7 days post-treatment. The efficacious treatments of HT-HaT-DOX were shown to induce a significant drop in sO2 (>10%) as early as 30 min post-treatment that led to tumor regression (in 90% of cases); HT-Saline and non-efficacious HT-HaT-DOX (10% of cases) treatments did not show any significant change in sO2 at these timepoints. The changes in sO2 were further corroborated with histological data, using the vascular and perfusion markers CD31 and FITC-lectin. These results allowed us to further surmise a plausible mechanism of the cellular events taking place in the TSL treated tumor regions over the first 24 hours post-treatment. The potential for using photoacoustic imaging to measure tumor sO2 as a surrogate prognostic marker for predicting therapeutic outcome with a TSL treatment is demonstrated.

High-frequency ultrasound analysis of post-mitotic arrest cell death.

Non-invasive monitoring of cancer cell death would permit rapid feedback on treatment response. One technique showing such promise is quantitative ultrasound. High-frequency ultrasound spectral radiofrequency analysis was used to study cell death in breast cancer cell samples. Quantitative ultrasound parameters, including attenuation, spectral slope, spectral 0-MHz-intercept, midband fit, and fitted parameters displayed significant changes with paclitaxel-induced cell death, corresponding to observations of morphological changes seen in histology and electron microscopy. In particular, a decrease in spectral slope from 0.24±0.07 dB/MHz to 0.04±0.09 dB/MHz occurred over 24 hours of treatment time and was identified as an ultrasound parameter capable of differentiating post-mitotic arrest cell death from classical apoptosis. The formation of condensed chromatin aggregates of 1 micron or greater in size increased the number of intracellular scatterers, consistent with a hypothesis that nuclear material is a primary source of ultrasound scattering in dying cells. It was demonstrated that the midband fit quantitatively correlated to cell death index, with a Pearson R-squared value of 0.99 at p<0.01. These results suggest that high-frequency ultrasound can not only qualitatively assess the degree of cancer cell death, but may be used to quantify the efficacy of chemotherapeutic treatments.

Quantitative Ultrasound Comparison of MAT and 4T1 Mammary Tumors in Mice and Rats Across Multiple Imaging Systems.

OBJECTIVES: Quantitative ultrasound estimates such as the frequency-dependent backscatter coefficient (BSC) have the potential to enhance noninvasive tissue characterization and to identify tumors better than traditional B-mode imaging. Thus, investigating system independence of BSC estimates from multiple imaging platforms is important for assessing their capabilities to detect tissue differences. METHODS: Mouse and rat mammary tumor models, 4T1 and MAT, respectively, were used in a comparative experiment using 3 imaging systems (Siemens, Ultrasonix, and VisualSonics) with 5 different transducers covering a range of ultrasonic frequencies. RESULTS: Functional analysis of variance of the MAT and 4T1 BSC-versus-frequency curves revealed statistically significant differences between the two tumor types. Variations also were found among results from different transducers, attributable to frequency range effects. At 3 to 8 MHz, tumor BSC functions using different systems showed no differences between tumor type, but at 10 to 20 MHz, there were differences between 4T1 and MAT tumors. Fitting an average spline model to the combined BSC estimates (3-22 MHz) demonstrated that the BSC differences between tumors increased with increasing frequency, with the greatest separation above 15 MHz. Confining the analysis to larger tumors resulted in better discrimination over a wider bandwidth. CONCLUSIONS: Confining the comparison to higher ultrasonic frequencies or larger tumor sizes allowed for separation of BSC-versus-frequency curves from 4T1 and MAT tumors. These constraints ensure that a greater fraction of the backscattered signals originated from within the tumor, thus demonstrating that statistically significant tumor differences were detected.

Techniques and evaluation from a cross-platform imaging comparison of quantitative ultrasound parameters in an in vivo rodent fibroadenoma model.

This contribution demonstrates that quantitative ultrasound (QUS) capabilities are platform independent, using an in vivo model. Frequency-dependent attenuation estimates, backscatter coefficient, and effective scatterer diameter estimates are shown to be comparable across four different ultrasound imaging systems with varied processing techniques. The backscatter coefficient (BSC) is a fundamental material property from which several QUS parameters are estimated; therefore, consistent BSC estimates among different systems must be demonstrated. This study is an intercomparison of BSC estimates acquired by three research groups (UIUC, UW, ISU) from four in vivo spontaneous rat mammary fibroadenomas using three clinical array systems and a single-element laboratory scanner system. Because of their highly variable backscatter properties, fibroadenomas provided an extreme test case for BSC analysis, and the comparison is across systems for each tumor, not across the highly heterogeneous tumors. RF echo data spanning the 1 to 12 MHz frequency range were acquired in three dimensions from all animals using each system. Each research group processed their RF data independently, and the resulting attenuation, BSC, and effective scatterer diameter (ESD) estimates were compared. The attenuation estimates across all systems showed the same trends and consistently fit the power-law dependence on frequency. BSCs varied among the multiple slices of data acquired by each transducer, with variations between transducers being of a similar magnitude as those from slice to slice. Variation between BSC estimates was assessed via functional signal-to-noise ratios derived from backscatter data. These functional signal-to-noise ratios indicated that BSC versus frequency variations between systems ranged from negligible compared with the noise level to roughly twice the noise level. The corresponding functional analysis of variance (fANOVA) indicated statistically significant differences between BSC curves from different systems. However, root mean squared difference errors of the BSC values (in decibels) between different transducers and imaging platforms were less than half of the BSC magnitudes in most cases. Statistical comparison of the effective scatterer diameter (ESD) estimates resulted in no significant differences in estimates from three of the four transducers used for those estimates, demonstrating agreement among estimates based on the BSC. This technical advance demonstrates that these in vivo measurements can be made in a system-independent manner; the necessary step toward clinical implementation of the technology.

Comparison of ultrasound attenuation and backscatter estimates in layered tissue-mimicking phantoms among three clinical scanners.

Backscatter and attenuation coefficient estimates are needed in many quantitative ultrasound strategies. In clinical applications, these parameters may not be easily obtained because of variations in scattering by tissues overlying a region of interest (ROI). The goal of this study is to assess the accuracy of backscatter and attenuation estimates for regions distal to nonuniform layers of tissue-mimicking materials. In addition, this work compares results of these estimates for "layered" phantoms scanned using different clinical ultrasound machines. Two tissue-mimicking phantoms were constructed, each exhibiting depth-dependent variations in attenuation or backscatter. The phantoms were scanned with three ultrasound imaging systems, acquiring radio frequency echo data for offline analysis. The attenuation coefficient and the backscatter coefficient (BSC) for sections of the phantoms were estimated using the reference phantom method. Properties of each layer were also measured with laboratory techniques on test samples manufactured during the construction of the phantom. Estimates of the attenuation coefficient versus frequency slope, α(0), using backscatter data from the different systems agreed to within 0.24 dB/cm-MHz. Bias in the α(0) estimates varied with the location of the ROI. BSC estimates for phantom sections whose locations ranged from 0 to 7 cm from the transducer agreed among the different systems and with theoretical predictions, with a mean bias error of 1.01 dB over the used bandwidths. This study demonstrates that attenuation and BSCs can be accurately estimated in layered inhomogeneous media using pulse-echo data from clinical imaging systems.

Cross-imaging system comparison of backscatter coefficient estimates from a tissue-mimicking material.

A key step toward implementing quantitative ultrasound techniques in a clinical setting is demonstrating that parameters such as the ultrasonic backscatter coefficient (BSC) can be accurately estimated independent of the clinical imaging system used. In previous studies, agreement in BSC estimates for well characterized phantoms was demonstrated across different laboratory systems. The goal of this study was to compare the BSC estimates of a tissue mimicking sample measured using four clinical scanners, each providing RF echo data in the 1-15 MHz frequency range. The sample was previously described and characterized with single-element transducer systems. Using a reference phantom for analysis, excellent quantitative agreement was observed across the four array-based imaging systems for BSC estimates. Additionally, the estimates from data acquired with the clinical systems agreed with theoretical predictions and with estimates from laboratory measurements using single-element transducers.

Ultrasonic attenuation and backscatter coefficient estimates of rodent-tumor-mimicking structures: comparison of results among clinical scanners.

In vivo estimations of the frequency-dependent acoustic attenuation (alpha) and backscatter (eta) coefficients using radiofrequency (rf) echoes acquired with clinical ultrasound systems must be independent of the data acquisition setup and the estimation procedures. In a recent in vivo assessment of these parameters in rodent mammary tumors, overall agreement was observed among alpha and eta estimates using data from four clinical imaging systems. In some cases, particularly in highly-attenuating heterogeneous tumors, multisystem variability was observed. This paper compares alpha and eta estimates of a well-characterized rodent-tumor-mimicking homogeneous phantom scanned using seven transducers with the same four clinical imaging systems: a Siemens Acuson S2000, an Ultrasonix RP, a Zonare Z.one and a VisualSonics Vevo2100. alpha and eta estimates of lesion-mimicking spheres in the phantom were independently assessed by three research groups, who analyzed their system's rf echo signals. Imaging-system-based estimates of alpha and eta of both lesion-mimicking spheres were comparable to through-transmission laboratory estimates and to predictions using Faran's theory, respectively. A few notable variations in results among the clinical systems were observed but the average and maximum percent difference between alpha estimates and laboratory-assessed values was 11% and 29%, respectively. Excluding a single outlier dataset, the average and maximum average difference between eta estimates for the clinical systems and values predicted from scattering theory was 16% and 33%, respectively. These results were an improvement over previous interlaboratory comparisons of attenuation and backscatter estimates. Although the standardization of our estimation methodologies can be further improved, this study validates our results from previous rodent breast-tumor model studies.

Ultrasonic backscatter coefficients for weakly scattering, agar spheres in agar phantoms.

Applicability of ultrasound phantoms to biological tissue has been limited because most phantoms have generally used strong scatterers. The objective was to develop very weakly scattering phantoms, whose acoustic scattering properties are likely closer to those of tissues and then compare theoretical simulations and experimental backscatter coefficient (BSC) results. The phantoms consisted of agar spheres of various diameters (nominally between 90 and 212 microm), containing ultrafiltered milk, suspended in an agar background. BSC estimates were performed at two institutions over the frequency range 1-13 MHz, and compared to three models. Excellent agreement was shown between the two laboratory results as well as with the three models.

Cross-imaging platform comparison of ultrasonic backscatter coefficient measurements of live rat tumors.

OBJECTIVE: To translate quantitative ultrasound (QUS) from the laboratory into the clinic, it is necessary to demonstrate that the measurements are platform independent. Because the backscatter coefficient (BSC) is the fundamental estimate from which additional QUS estimates are calculated, agreement between BSC results using different systems must be demonstrated. This study was an intercomparison of BSCs from in vivo spontaneous rat mammary tumors acquired by different groups using 3 clinical array systems and a single-element laboratory scanner system. METHODS: Radio frequency data spanning the 1- to 14-MHz frequency range were acquired in 3 dimensions from all animals using each system. Each group processed their radio frequency data independently, and the resulting BSCs were compared. The rat tumors were diagnosed as either carcinoma or fibroadenoma. RESULTS: Carcinoma BSC results exhibited small variations between the multiple slices acquired with each transducer, with similar slopes of BSC versus frequency for all systems. Somewhat larger variations were observed in fibroadenomas, although BSC variations between slices of the same tumor were of comparable magnitude to variations between transducers and systems. The root mean squared (RMS) errors between different transducers and imaging platforms were highly variable. The lowest RMS errors were observed for the fibroadenomas between 4 and 5 MHz, with an average RMS error of 4 x 10(-5) cm(-1)Sr(-1) and an average BSC value of 7.1 x 10(-4) cm(-1)Sr(-1), or approximately 5% error. The highest errors were observed for the carcinoma between 7 and 8 MHz, with an RMS error of 1.1 x 10(-1) cm(-1)Sr(-1) and an average BSC value of 3.5 x 10(-2) cm(-1)Sr(-1), or approximately 300% error. CONCLUSIONS: This technical advance shows the potential for QUS technology to function with different imaging platforms.

A new three-dimensional ultrasound microimaging technology for preclinical studies using a transgenic prostate cancer mouse model.

Prostate cancer is the most common cancer in adult men in North America. Preclinical studies of prostate cancer employ genetically engineered mouse models, because prostate cancer does not occur naturally in rodents. Widespread application of these models has been limited because autopsy was the only reliable method to evaluate treatment efficacy in longitudinal studies. This article reports the first use of three-dimensional ultrasound microimaging for measuring tumor progression in a genetically engineered mouse model, the 94-amino acid prostate secretory protein gene-directed transgenic prostate cancer model. Qualitative comparisons of three-dimensional ultrasound images with serial histology sections of prostate tumors show the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. Ultrasound imaging identified tumors ranging from 2.4 to 14 mm maximum diameter. The correlation coefficient of tumor diameter measurements done in vivo with three-dimensional ultrasound and at autopsy was 0.998. Prospective tumor detection sensitivity and specificity were both >90% when diagnoses were based on repeated ultrasound examinations done on separate days. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated volume doubling times of 5 and 13 days for two prostate tumors. Compared with other microimaging and molecular imaging modalities, the application of three-dimensional ultrasound imaging to prostate cancer in mice showed advantages, such as high spatial resolution and contrast in soft tissue, fast and uncomplicated protocols, and portable and economical equipment that will likely enable ultrasound to become a new microimaging modality for mouse preclinical trial studies.

Three-dimensional high-frequency ultrasound imaging for longitudinal evaluation of liver metastases in preclinical models.

Liver metastasis is a clinically significant contributor to the mortality associated with melanoma, colon, and breast cancer. Preclinical mouse models are essential to the study of liver metastasis, yet their utility has been limited by the inability to study this dynamic process in a noninvasive and longitudinal manner. This study shows that three-dimensional high-frequency ultrasound can be used to noninvasively track the growth of liver metastases and evaluate potential chemotherapeutics in experimental liver metastasis models. Liver metastases produced by mesenteric vein injection of B16F1 (murine melanoma), PAP2 (murine H-ras-transformed fibroblast), HT-29 (human colon carcinoma), and MDA-MB-435/HAL (human breast carcinoma) cells were identified and tracked longitudinally. Tumor size and location were verified by histologic evaluation. Tumor volumes were calculated from the three-dimensional volumetric data, with individual liver metastases showing exponential growth. The importance of volumetric imaging to reduce uncertainty in tumor volume measurement was shown by comparing three-dimensional segmented volumes with volumes estimated from diameter measurements and the assumption of an ellipsoid shape. The utility of high-frequency ultrasound imaging in the evaluation of therapeutic interventions was established with a doxorubicin treatment trial. These results show that three-dimensional high-frequency ultrasound imaging may be particularly well suited for the quantitative assessment of metastatic progression and the evaluation of chemotherapeutics in preclinical liver metastasis models.