Sebastian Brand

Quantitative Ultrasound Characterization of Responses to Radiotherapy in Cancer Mouse Models

Purpose: Currently, no imaging modality is used routinely to assess tumor responses to radiotherapy within hours to days after the delivery of treatment. In this study, we show the application of quantitative ultrasound methods to characterize tumor responses to cancer radiotherapy in vivo, as early as 24 hours after treatment administration. Experimental Design: Three mouse models of head and neck cancer were exposed to radiation doses of 0, 2, 4, and 8 Gray. Data were collected with an ultrasound scanner using frequencies of 10 to 30 MHz. Ultrasound estimates calculated from normalized power spectra and parametric images (spatial maps of local estimates of ultrasound parameters) were used as indicators of response. Results: Two of the mouse models (FaDu and C666-1) exhibited large hyperechoic regions at 24 hours after radiotherapy. The ultrasound integrated backscatter increased by 6.5 to 8.2 dB (P < 0.001) and the spectral slopes increased from 0.77 to 0.90 dB/MHz for the C666-1 tumors and from 0.54 to 0.78 dB/MHz for the FaDu tumors (P < 0.05), in these regions compared with preirradiated tumors. The hyperechoic regions in the ultrasound images corresponded in histology to areas of cell death. Parametric images could discern the tumor regions that responded to treatment. The other cancer mouse model (Hep-2) was resistant to radiotherapy. Conclusions: The results indicate that cell structural changes after radiotherapy have a significant influence on ultrasound spectral parameters. This provides a foundation for future investigations regarding the use of ultrasound in cancer patients to individualize treatments noninvasively based on their responses to specific interventions.

HIGH FREQUENCY ULTRASOUND TISSUE CHARACTERIZATION AND ACOUSTIC MICROSCOPY OF INTRACELLULAR CHANGES

The objective of this work is to investigate changes in the acoustic properties of cells when exposed to chemotherapy for monitoring treatment response. High frequency ultrasound spectroscopy (10-60 MHz) and scanning acoustic microscopy (0.9 GHz) were performed on HeLa cells (Ackermann et al. 1954, Masters 2002) that were exposed to the chemotherapeutic agent cisplatin. Ultrasonic radio-frequency data were acquired from pellets containing HeLa cells after exposure to cisplatin to induce apoptosis. Scanning acoustic and laser fluorescence microscopy images were recorded from single HeLa cells exposed to the same drug. Data acquisition in both cases was performed at several time points throughout the chemotherapeutic treatment for up to 27 h. In the high frequency ultrasound investigation, normalized power spectra were calculated within a region-of-interest. A 20 MHz transducer (f-number 2.35) and a 40 MHz transducer (f-number 3) were used for the data collection in the high frequency ultrasound experiments. The backscatter coefficients, integrated backscatter coefficients, mid-band fit and spectral slope were computed as a function of treatment time to monitor acoustical property changes during apoptosis. Acoustic attenuation was measured using the spectral substitution technique at all time points. Spectral parameter changes were detected after 12 h of exposure and coincided with the initiation of cell damage as assessed by optical microscopy. Integrated backscatter coefficients increased by over 100% between 0 h and 24 h of treatment, with small changes in the associated attenuation ( approximately 0.1 dB/[MHz cm]). Acoustic microscopy was performed at 0.9 GHz frequency. The cell structure was imaged using staining in laser fluorescence microscopy. All cells showed excellent correspondence between the locations of apoptotic nuclear condensation observed in optical imaging and changes in attenuation contrast in acoustic microscopy images. The time after drug exposure at which such changes occurred in the optical images were coincident with the time of changes detected in the acoustic microscopy images and the high frequency ultrasound experiments.

Automated inspection and classification of flip-chip-contacts using scanning acoustic microscopy

Industrial applications often require failure analysis methods working non-destructively, enabling either a rapid quality control or fault isolation and defect localization prior to a detailed defect investigation requiring target preparation. Scanning acoustic microscopy in the frequency range above 100 MHz provides high axial and lateral resolution, a moderate penetration depth and the required non-destructivity. In this study a method for an automated detection of defects in flip-chip-contacts was developed. Chip samples were manufactured in flip-chip technology containing a 750 μm thick die with solder balls (80 μm diameter) and underfill attached to an organic-layer substrate. For acoustic inspection a scanning acoustic microscope in combination with a 175 MHz transducer was used. Recorded echo signals were analyzed off-line applying custom-made MATLAB software. For differentiation between the flip-chip-contacts and the underfill, the recorded echo signals were pre-analyzed. Signals obtained from the contacts were then inspected by wavelet-, pulse separation- and backscatter amplitude integral analysis. Complementary X-ray- and SEM-inspection was performed for defect verification. The separation of pulses obtained from the interfaces of the contacts, the absolute values and the distribution of wavelet coefficients corresponded to the interconnecting condition. The success rate of detecting voids was 96.8% as verified by SEM-imaging, while manual X-ray inspection showed success only in 64% of the analysed cases.

Ultrasound velocity and attenuation of porcine soft tissues with respect to structure and composition: I. Muscle

Ultrasound velocity and attenuation of soft tissues have been widely investigated. However, few studies completely covered considerable variations of both, structure and composition. The aim of this study was to collect acoustic reference data of porcine Longissimus muscle and associate them with compositional traits. In addition, measurements were conducted on fresh, formalin fixed, and frozen-thawed samples to evaluate the effect of processing on ultrasound parameters and comparisons with earlier investigations. Measurement conditions (temperature and fibre orientation) were realised close to hanging carcasses conditions. Sound velocity ranged from 1617 ± 6 to 1622 ± 5 ms(-1), while attenuation mostly ranged from 1.0 ± 0.3 to 1.2 ± 0.3 dB MHz(-1)cm(-1). Only formalin fixed samples showed significantly higher attenuation (2.2 ± 0.6 dB MHz(-1)cm(-1)). Highest correlations have been observed between intramuscular fat and attenuation (up to r = .7). The obtained results are anticipated to improve ultrasound based estimation of the intramuscular fat of pig muscle on intact carcasses.

Monitoring of cell death in epithelial cells using high frequency ultrasound spectroscopy.

Spectral and wavelet analyses were performed on ultrasound radiofrequency (RF) data collected from centrifuged cell samples containing HEp-2 cells after induction of apoptosis by exposure to camptothecin. Samples were imaged at several time points after drug exposure using high-frequency ultrasound in the range from 10-60 MHz. A 20-MHz transducer with a f-number of 2.35 and a 40-MHz transducer with a f-number of 3 were used for collecting the RF data. Normalized power spectra were computed from the backscattered ultrasound signals within a region-of-interest (ROI) for further analysis. Spectral slopes, integrated backscatter coefficients (IBCs) and wavelet parameters were estimated as a function of treatment time to monitor acoustic property changes during apoptosis. Changes in spectral parameters were detected starting six hours after treatment and coincided with changes in corresponding histology. Throughout the course of chemotherapy, variation in estimates of the spectral slope of up to 35% were observed. During the treatment, IBCs increased by 400% compared with estimates obtained from the control samples. Changes in spectral parameters are hypothesized to be linked to structural cell changes during apoptosis. In addition, the sensitivity of a wavelet-based analysis to the ultrasonic assessment of cellular changes was investigated. Results of the wavelet analysis showed variations similar to the spectral parameters. Where values of the spectral slope decreased, estimates of the scaling factors increased. Because wavelet analysis preserves the signal-time localization, its application will be potentially beneficial for assessing treatment responses in vivo. The current study contributes toward the development of a non-invasive method for monitoring apoptosis as a measure of the success of chemotherapeutic treatment of cancer.

Ultrasound velocity and attenuation of porcine soft tissues with respect to structure and composition: II. Skin and backfat

Ultrasound is regarded as a promising method to determine the intramuscular fat content of pork loin. At intact carcasses, the signal passes the backfat whose ultrasound parameters (sound velocity and attenuation) have not been fully investigated. This study intended to collect a dataset of ultrasound parameters for individual backfat layers and to elucidate relationships with structural and compositional characteristics. In-vitro measurements at 10 MHz were conducted on backfat samples of pork carcasses representative for German populations. The average sound velocity ranged from 1436 ± 9 to 1470 ± 37 ms(-1) for the fat layers, and 1682 ± 23 ms(-1) for skin. Velocity of the compound backfat decreased with overall thickness. Attenuation was not affected by thickness ranging between 1.6 ± 0.7 and 2.7 ± 1.5 dB MHz(-1)cm(-1) for all layers. Sound velocity was negatively correlated with fat content and dry matter. The obtained results are anticipated to improve signal correction prior to spectral analysis of ultrasound measurements at intact carcasses.

Prediction of the intramuscular fat content in loin muscle of pig carcasses by quantitative time-resolved ultrasound.

A novel method for non-destructive intramuscular fat (IMF) estimation via spectral ultrasound backscatter analysis of signals obtained from pig carcasses early post mortem is described. A commercial hand-held ultrasound device (center frequency: 2.7 MHz) was modified to focus the sound beam to the longissimus muscle at the 2nd/3rd last rib. Time-resolved ultrasound backscatter signals of loin muscle were recorded 45 min p.m. on 82 pig carcass sides. Backfat width (d(BF)=18.9±3.8 mm) and muscle attenuation (α(muscle)=.77±.15 dB MHz(-1) cm(-1)) were assessed from the measured pulse echo data. Other propagation properties of skin, backfat and muscle tissue obtained in a previous investigation were incorporated into the signal pre-processing to minimize parameter estimation artifacts. Spectral and cepstral parameters were derived from time-gated backscattered signals measured in the central muscle region. The range of intramuscular fat (IMF) determined by ether extraction was representative for German pig populations (.7%≤IMF(chem)≤3.6%, coefficient of variation CV(IMF(chem))=44.8%). Variations of IMF were associated with variations of backfat width (CV(d(BF))=20.2%), muscle attenuation (CV(α(muscle))=19.3%), and slope of the backscattered amplitude spectrum (CV(m)=28.8%). A full cross validated multiple linear regression model using these parameters resulted in good predictability of IMF(chem) (R(2)=.76, RMSEP=.34%). Among all tested carcasses, 73% could be correctly classified into one of three IMF classes (LOW: <1%, MID: 1-2%, HIGH: >2%). Using a single threshold (2% IMF), about 92% of all carcasses were correctly classified. With respect to the inherent variability of IMF within a single muscle and the different tissue volumes used for the chemical and ultrasound based IMF estimations the remaining prediction errors are acceptable. Compared to previous ultrasound based studies, the number of acoustic parameters used for the IMF prediction could be reduced. Moreover, the used parameters are based on time-of-flight and spectral slope estimations, which are i) more robust with respect to measurement artifacts and ii) have a causal link to structural variations associated with IMF variations in pork loin.

High frequency scanning acoustic microscopy applied to 3D integrated process: Void detection in Through Silicon Vias

Among the technological developments pushed by the emergence of 3D-ICs, Through Silicon Via (TSV) technology has become a standard element in device processing over the past years. As volume increases, defect detection in the overall TSV formation sequence is becoming a major element of focus nowadays. Robust methods for in-line void detection during TSV processing are therefore needed especially for scaled down dimensions. Within this framework, the current contribution describes the successful application of innovative GHz Scanning Acoustic Microscopy (SAM) to TSV void detection in a via-middle approach.

Scanning acoustic gigahertz microscopy for metrology applications in three-dimensional integration technologies

Abstract. Current trends in microelectronics focus on three-dimensionally integrating different components to allow for increasing density and functionality of integrated systems. Concepts pursued involve vertical stacking and interconnecting technologies that employ micro bumping, wafer bonding, and through silicon vias (TSVs). Both the increasing complexity and the miniaturization of key elements in three-dimensional (3-D) components lead to new requirements on inspection and metrology tools and techniques as well as for failure analysis methodologies. For metrology and quality assessment in particular, methods operating nondestructively are of major importance. Scanning acoustic microscopy has the ability of illuminating optically opaque materials and, thus, allowing the assessment and imaging of internal structures. Conventional scanning acoustic microscopy (SAM) equipment can be applied to analyze the quality of wafer-bonded interfaces in 3-D integration but may reach its limitations when structures shrink in size and gain complexity. A new concept of acoustic inspection in the gigahertz (GHz) frequency band is explored for its applicability to 3-D integration technologies. Extending the acoustic inspection frequency allows for lateral resolutions in the 1-μm range and also enables the inspection of microbumps and TSVs in addition to wafer bonded interfaces, which exceed the applicability of conventional SAM. Three case studies are presented here ranging from conventional SAM on a full wafer scale to acoustic GHz microscopy on thin films and TSVs.

Ceramic substrates with aluminum metallization for power application

The reliability of power electronic devices is significantly related to the material properties of the applied substrates which carry the semiconductor chip and the electric interconnections. The most common solution to fulfill the stringent requirements of these devices, with respect to high isolation voltage, good thermal conductivity, high temperature cycling reliability and low cost, is to use ceramic substrates with copper layers on both sides. However, the currently increasing reliability standards in power electronics lead to a situation where common DCB substrates reach their limits in meeting these higher requirements.