Elizabeth S. L. Berndl

Probing red blood cell morphology using high-frequency photoacoustics.

A method that can rapidly quantify variations in the morphology of single red blood cells (RBCs) using light and sound is presented. When irradiated with a laser pulse, an RBC absorbs the optical energy and emits an ultrasonic pressure wave called a photoacoustic wave. The power spectrum of the resulting photoacoustic wave contains distinctive features that can be used to identify the RBC size and morphology. When particles 5-10 μm in diameter (such as RBCs) are probed with high-frequency photoacoustics, unique periodically varying minima and maxima occur throughout the photoacoustic signal power spectrum at frequencies >100 MHz. The location and distance between spectral minima scale with the size and morphology of the RBC; these shifts can be used to quantify small changes in the morphology of RBCs. Morphological deviations from the normal biconcave RBC shape are commonly associated with disease or infection. Using a single wide-bandwidth transducer sensitive to frequencies between 100 and 500 MHz, we were able to differentiate healthy RBCs from irregularly shaped RBCs (such as echinocytes, spherocytes, and swollen RBCs) with high confidence using a sample size of just 21 RBCs. As each measurement takes only seconds, these methods could eventually be translated to an automated device for rapid characterization of RBC morphology and deployed in a clinical setting to help diagnose RBC pathology.

High frequency label-free photoacoustic microscopy of single cells

Photoacoustic measurements of melanoma cells and red blood cells (RBCs) using ultra-high frequency (UHF) wide-bandwidth transducers are reported. In this detection system, the resolution typically depends on the parameters of the receiving transducer, and not the focus of the laser. A single melanoma cell was imaged with 200, 375 and 1200 MHz transducers. As the frequency increased, the resolution increased, resulting in greater detail observed. A single RBC was imaged at 1200 MHz, showing the contours of the cell. While lateral and axial resolutions approaching 1 μm are possible with this microscope, the key advantage is the ability to perform a wide-bandwidth quantitative signal analysis of the photoacoustic signals. The power spectrum of the signals measured from RBCs showed distinct spectral minima around 800 and 1500 MHz which are directly related to the RBC geometry. This study reports on the high-resolution imaging capabilities and quantitative analyses using UHF photoacoustic microscopy.

High-Frequency Acoustic Impedance Imaging of Cancer Cells.

Variations in the acoustic impedance throughout cells and tissue can be used to gain insight into cellular microstructures and the physiologic state of the cell. Ultrasound imaging can be used to create a map of the acoustic impedance, on which fluctuations can be used to help identify the dominant ultrasound scattering source in cells, providing information for ultrasound tissue characterization. The physiologic state of a cell can be inferred from the average acoustic impedance values, as many cellular physiologic changes are linked to an alteration in their mechanical properties. A recently proposed method, acoustic impedance imaging, has been used to measure the acoustic impedance maps of biological tissues, but the method has not been used to characterize individual cells. Using this method to image cells can result in more precise acoustic impedance maps of cells than obtained previously using time-resolved acoustic microscopy. We employed an acoustic microscope using a transducer with a center frequency of 375 MHz to calculate the acoustic impedance of normal (MCF-10 A) and cancerous (MCF-7) breast cells. The generated acoustic impedance maps and simulations suggest that the position of the nucleus with respect to the polystyrene substrate may have an effect on the measured acoustic impedance value of the cell. Fluorescence microscopy and confocal microscopy were used to correlate acoustic impedance images with the position of the nucleus within the cell. The average acoustic impedance statistically differed between normal and cancerous breast cells (1.636 ± 0.010 MRayl vs. 1.612 ± 0.006 MRayl), indicating that acoustic impedance could be used to differentiate between normal and cancerous cells.

Properties of cells through life and death – an acoustic microscopy investigation

Current methods to evaluate the status of a cell are largely focused on fluorescent identification of molecular biomarkers. The invasive nature of these methods – requiring either fixation, chemical dyes, genetic alteration, or a combination of these – prevents subsequent analysis of samples. In light of this limitation, studies have considered the use of physical markers to differentiate cell stages. Acoustic microscopy is an ultrahigh frequency (>100 MHz) ultrasound technology that can be used to calculate the mechanical and physical properties of biological cells in real-time, thereby evaluating cell stage in live cells without invasive biomarker evaluation. Using acoustic microscopy, MCF-7 human breast adenocarcinoma cells within the G1, G2, and metaphase phases of the proliferative cell cycle, in addition to early and late programmed cell death, were examined. Physical properties calculated include the cell height, sound speed, acoustic impedance, cell density, adiabatic bulk modulus, and the ultrasonic attenuation. A total of 290 cells were measured, 58 from each cell phase, assessed using fluorescent and phase contrast microscopy. Cells actively progressing from G1 to metaphase were marked by a 28% decrease in attenuation, in contrast to the induction of apoptosis from G1, which was marked by a significant 81% increase in attenuation. Furthermore late apoptotic cells separated into 2 distinct groups based on ultrasound attenuation, suggesting that presently-unidentified sub-stages may exist within late apoptosis. A methodology has been implemented for the identification of cell stages without the use of chemical dyes, fixation, or genetic manipulation.

Quantitative ultrasound analyses of cell starvation in HT-29 pellets

Spectral analysis of ultrasound backscatter data has been shown to be sensitive to cellular changes, particularly due to apoptosis. Different therapies that seek to destroy cells work activate different cell death mechanisms, most commonly apoptosis, but also oncosis or ischemic death. Pellets of HT-29 colon adenocarcinoma cells were placed in PBS at room temperature over the course of 56 hours and were imaged with high-frequency (55 MHz) ultrasound and the raw RF data processed. Due to the lack of nutrients available to the cells, they underwent oncosis. Attenuation slope, speed of sound (SOS), spectral slope and midband fit (MBF) were estimated every 8 hours over the course of 56 hours to determine changes due to starvation. The spectral slope decreased through time, with an increase at 40 hours. Midband fit and attenuation both showed a trend through time with three phases with the first significant increase at 16 hours and a second significant increase at 40 hours. Speed of sound increased from 1513 m/s to 1534 m/s with time. A decrease in cellular number density and increase in inter-cellular debris is evident on histological slides. Results show different trends than observed for cells undergoing apoptosis, suggesting there are specific signatures that can be exploited to determine changes in cell morphology associated with different mechanisms of cell death.

Ultrasonic characterization of extra-cellular matrix in decellularized murine kidney and liver

Three-dimensional scaffolds are essential to the field of tissue engineering. While novel synthetic structures are being developed, there is still a great interest in exploring natural scaffolds in tissue, the extra-cellular matrix (ECM). A recently developed technique known as “decellularizing” allows for the removal of cells from intact tissue while preserving the ECM structure. In order to exploit the uniqueness of the native ECM, a structure which varies significantly between organs, it first needs to be well studied. This study outlines the use of quantitative ultrasound as a non-destructive method to characterize the extracellular matrix of excised murine kidneys and livers. This allows for the study of both natural tissue scaffolds, as well as the contributions of the cellular and extra-cellular components to ultrasound backscatter. In this study, excised murine livers and kidneys were imaged with a VisualSonics Vevo2100 using nominal 40 MHz linear-array transducer, after being maintained in PBS. Subsequently the organs were decellularized, in this process, the ECM of the tissue is isolated from its inhabiting cells, leaving an ECM scaffold of the tissue. The remaining extracellular matrix structures were reimaged. Raw RF data was acquired and normalized by a reference phantom. Linear fits to the normalized power spectra allow for the estimation and comparison of the spectral slope and midband fit. After being decellularized, the organs were significantly smaller in volume with increased backscatter in the liver and overall decrease in the kidney. The heterogeneous structure of the kidney was apparent in parametric images, with the spectral slope and midband fit higher in the central medulla region. The ability to compare backscatter from the extracellular matrix with and without cells allows for a detailed analysis of the contribution of individual cells to the ultrasound backscatter and could be employed to evaluate scaffold structures and progress of growth on these scaffolds.

Mean scatterer spacing estimation from pellets using cepstral analysis: A preliminary study

Ultrasonic backscattered signals from biological tissues contain information regarding their structures, more specifically, their scatterer structures. This work investigates the use of cepstral analysis in characterizing periodicities in ultrasound Alines due to uniformity in the scatterer distribution. The A-line is simulated as a convolution between a generated radio-frequency (RF) pulse and a scattering medium containing uniformly distributed scatterers along with randomly situated scatterers. The cepstral analysis is tested by varying the regularity of the scatterers. Simulation results indicate that the mean scatterer spacing can be estimated using both power and complex cepstrum, where it manifests itself as a peak. Experimental results conducted on cell pellets imaged at 1 and 56 hrs revealed a peak at 0.15 mm. In conclusion, a general agreement between simulation and experimental results was found. Future work include the further investigation of the use of mean scatterer spacing as a new biomarker for cancer treatment monitoring.

Acoustic and photoacoustic imaging of spheroids

Acoustic and photoacoustic high frequency imaging (50-100 MHz) can be used to generate images of cell constructs and spheroids with good spatial resolution and contrast. Here we demonstrate how co-registered acoustic and photoacoustic imaging can be used for imaging spheroids. Spheroids are widely used in cancer research and biology since they emulate a 3-dimensional environment such as that experienced in tumors. Spheroids were made by the hanging-drop method using the MCF-7 cancer cell line. To generate photoacoustic contrast, MCF-7 cells were incubated with optical absorbing nanoparticles (e.g. gold nanorods, 780nm absorption) for 24 hours and mixed with native MCF-7 cells prior to spheroid formation. The spheroids were between 0.5 mm and 1mm in diameter. Imaging was performed with the VisualSonics VEVO 770 (25-55 MHz) and a high-resolution SASAM acoustic/photoacoustic microscope for frequencies over 80 MHz (Kibero GmbH, Germany). The spheroid was imaged first using pulse echo ultrasound, then with pho...

Differentiation between cellularized and decellularized mouse kidneys using mean scatterer spacing: A preliminary study

Scattering from the extracellular matrix (ECM) is currently being investigated, using a decellularization technique, which involves removing cells from tissue while preserving the ECM. This work aims to investigate the use of the mean scatterer spacing, using cepstral analysis techniques, for the differentiation between cellularized and decellularized mouse kidneys. After decellularization, the mean scatterer spacing decreased, with an average spacing for all the kidneys of 5.97 ± 1.89 μm before decellularization, and 5.38 ± 1.72 μm after decellularization. A significant difference was found between the calculated spacings from the kidneys, before and after decellularization. Future work include the incorporation of other parameters to further improve the sensitivity of this technique.

Effective scatterer size estimates in HT-29 spheroids at 55 MHz and 80 MHz

Spheroids provide a three-dimensional in-vitro model of cell-to-cell interaction and basic structure. High-frequency ultrasound studies of spheroids show good image contrast between viable rims and necrotic cores, but spectral analyses have not been performed. This study examines colorectal HT-29 spheroids using high-frequency ultrasound at 55MHz (n=5) and 80MHz (n=4) to determine changes in spectral parameters as a function of transducer frequency and location (core vs rim). Histology shows low cell density within the core and a tightly packed rim, which forms a smooth shell. Statistically significant differences were found in the midband fit, spectral slope and effective scatterer diameter estimate as a function of imaging frequency and spheroid location. Effective scatterer diameter estimates (12.7 ± 0.4 μm at 55MHz and 6.6 ± 0.5 μm at 80MHz) agree closely with histological estimates (12.9 ± 0.3 μm for the cell and 7.0 ± 0.3 μm for the nucleus) suggesting different primary scatterers at the two frequencies.