Noninvasive Quantification of Contractile Dynamics in Cardiac Cells, Spheroids, and Organs-on-a-Chip Using High-Frequency Ultrasound.

Cell-based models that mimic in vivo heart physiology are poised to make significant advances in cardiac disease modeling and drug discovery. In these systems, cardiomyocyte (CM) contractility is an important functional metric, but current measurement methods are inaccurate and low-throughput or require complex setups. To address this need, we developed a standalone noninvasive, label-free ultrasound technique operating at 40-200 MHz to measure the contractile kinetics of cardiac models, ranging from single adult CMs to 3D microtissue constructs in standard cell culture formats. The high temporal resolution of 1000 fps resolved the beat profile of single mouse CMs paced at up to 9 Hz, revealing limitations of lower speed optical based measurements to resolve beat kinetics or characterize aberrant beats. Coupling of ultrasound with traction force microscopy enabled the measurement of the CM longitudinal modulus and facile estimation of adult mouse CM contractile forces of 2.34 ± 1.40 µN, comparable to more complex measurement techniques. Similarly, the beat rate, rhythm, and drug responses of CM spheroid and microtissue models were measured, including in configurations without optical access. In conclusion, ultrasound can be used for the rapid characterization of CM contractile function in a wide range of commonly studied configurations ranging from single cells to 3D tissue constructs using standard well plates and custom microdevices, with applications in cardiac drug discovery and cardiotoxicity evaluation.

Air-Coupled Photoacoustic Detection of Airborne Particulates.

In this study, we present a novel method to detect airborne particulates using air-coupled photoacoustics, with a goal toward detecting viral content in respiratory droplets. The peak photoacoustic frequency emitted from micrometer-sized particulates is over 1000 MHz, but at this frequency, the signals are highly attenuated in air. Measurements were taken using a thin planar absorber and ultrasound transducers with peak sensitivity between 50 kHz and 2000 kHz and a 532 nm pulsed laser to determine the optimum detection frequency. 350 kHz to 500 kHz provided the highest amplitude signal while minimizing attenuation in air. To simulate the expulsion of respiratory droplets, an atomizer device was used to spray droplets into open air through a pulsed laser. Droplets were composed of water, water with acridine orange dye, and water with gold nanoparticles. The dye and nanoparticles were chosen due to their similarity in the UV absorption peaks when compared to RNA. Using a 260 nm laser, the average photoacoustic signal from water was the highest, and then the signal decreased with dye or nanoparticles. Increasing absorber concentrations within their respective solutions resulted in a decreasing photoacoustic signal, which is opposite to our expectations. Monte Carlo simulations demonstrated that depending on the droplet dimensions, water droplets focus photons to create a localized fluence elevation. Absorbers within the droplet can inhibit photon travel through the droplet, decreasing the fluence. Photoacoustic signals are created through optical absorption within the droplet, potentially amplified with the localized fluence increase through the droplet focusing effect, with a trade-off in signal amplitude depending on the absorber concentration.

Assessing engineered tissues and biomaterials using ultrasound imaging: In vitro and in vivo applications.

Quantitative assessment of the structural, functional, and mechanical properties of engineered tissues and biomaterials is fundamental to their development for regenerative medicine applications. Ultrasound (US) imaging is a non-invasive, non-destructive, and cost-effective technique capable of longitudinal and quantitative monitoring of tissue structure and function across centimeter to sub-micron length scales. Here we present the fundamentals of US to contextualize its application for the assessment of biomaterials and engineered tissues, both in vivo and in vitro. We review key studies that demonstrate the versatility and broad capabilities of US for clinical and pre-clinical biomaterials research. Finally, we highlight emerging techniques that further extend the applications of US, including for ultrafast imaging of biomaterials and engineered tissues in vivo and functional monitoring of stem cells, organoids, and organ-on-a-chip systems in vitro.

High-frequency quantitative ultrasound for the assessment of the acoustic properties of engineered tissues in vitro.

Acoustic properties of biomaterials and engineered tissues reflect their structure and cellularity. High-frequency ultrasound (US) can non-invasively characterize and monitor these properties with sub-millimetre resolution. We present an approach to estimate the speed of sound, acoustic impedance, and acoustic attenuation of cell-laden hydrogels that accounts for frequency-dependent effects of attenuation in coupling media, hydrogel thickness, and interfacial transmission/reflection coefficients of US waves, all of which can bias attenuation estimates. Cell-seeded fibrin hydrogel disks were raster-scanned using a 40 MHz US transducer. Thickness, speed of sound, acoustic impedance, and acoustic attenuation coefficients were determined from the difference in the time-of-flight and ratios of the magnitudes of US signals, interfacial transmission/reflection coefficients, and acoustic properties of the coupling media. With this approach, hydrogel thickness was accurately measured by US, with agreement to confocal microscopy (r2 = 0.97). Accurate thickness measurement enabled acoustic property measurements that were independent of hydrogel thickness, despite up to 60% reduction in thickness due to cell-mediated contraction. Notably, acoustic attenuation coefficients increased with increasing cell concentration (p < 0.001), reflecting hydrogel cellularity independent of contracted hydrogel thickness. This approach enables accurate measurement of the intrinsic acoustic properties of biomaterials and engineered tissues to provide new insights into their structure and cellularity. STATEMENT OF SIGNIFICANCE: High-frequency ultrasound can measure the acoustic properties of engineered tissues non-invasively and non-destructively with µm-scale resolution. Acoustic properties, including acoustic attenuation, are related to intrinsic material properties, such as scatterer density. We developed an analytical approach to estimate the acoustic properties of cell-laden hydrogels that accounts for the frequency-dependent effects of attenuation in coupling media, the reflection/transmission of ultrasound waves at the coupling interfaces, and the dependency of measurements on hydrogel thickness. Despite up to 60% reduction in hydrogel thickness due to cell-mediated contraction, our approach enabled measurements of acoustic properties that were substantially independent of thickness. Acoustic attenuation increased significantly with increasing cell concentration (p < 0.001), demonstrating the ability of acoustic attenuation to reflect intrinsic physical properties of engineered tissues.

Thrombin-responsive engineered nanoexcavator with full-thickness infiltration capability for pharmaceutical-free deep venous thrombosis theranostics.

Although nanotechnology has shown great promise for treating multiple vascular diseases in recent years, simultaneous noninvasive detection and efficient dissolution of deep venous thrombosis (DVT) still remains challenging. In particular, long blockage areas and large thrombus thicknesses in DVT cause enormous difficulties for site-specific deep-seated thrombus theranostics. Therefore, based on the unique components of DVT, the novel concept of a thrombin-responsive full-thickness infiltration nonpharmaceutical nanoplatform for DVT theranostics is proposed here. The penetration depth is innovatively enhanced with efficient targeting and accumulation in the whole thrombi. Herein, we report a thrombin-responsive phase-transition liposome incorporating a liquid perfluoropentane (PFP) core and modified with two binding peptides, activatable cell-penetrating peptide (ACPP) and fibrin-binding ligand (FTP), which contribute to efficient liposome targeting and accumulation within the thrombi. This targeted nanoplatform is constructed to dig out the thrombus with the assistance of low-intensity focused ultrasound (LIFU), performing the destructive function of an excavator via an acoustic droplet vaporization effect (acting as a "nanoexcavator" system), which can activate and vaporize into microbubbles to enhance LIFU efficacy. The resulting microbubbles enable real-time monitoring of the therapeutic process with ultrasound imaging and high performance photoacoustic imaging after loading DIR. This non-invasive nonpharmaceutical thrombolytic strategy is an improvement over existing clinical methods without systemic side effects.

Optical and photoacoustic radiofrequency spectroscopic analysis for detecting red blood cell death.

Under stress, red blood cells (RBCs) undergo programmed cell death (eryptosis). One of the signaling molecules for eryptosis, sphingomyelinase (SMase), plays an important role in monitoring the efficacy of vascular targeted cancer therapy. The high optical absorption of erythrocytes coupled with the changes of eryptotic RBCs makes RBCs ideal targets for the photoacoustic (PA) detection and characterization of vascular treatments. In this work, experiments characterizing eryptosis were performed: PA detection of high frequencies (>100 MHz) that enabled analysis at the single-cell level and of low frequencies (21 MHz) that enabled analysis at the RBC ensemble level. Ultrasound spectral analysis was performed on control and SMase-treated RBCs. Spectral unmixing was applied to quantify methemoglobin production as a by-product of RBC death. Validation was performed using a blood gas analyzer and optical spectrometry. Our results indicate that PA radiofrequency spectra could be used to differentiate the biochemically induced morphological changes as RBCs lose their native biconcave shape, and release hemoglobin into the surroundings. Spectral unmixing revealed a 7% increase in methemoglobin content for SMase-treated samples due to the oxidative stress on the RBCs. These findings suggest that PA spectral analysis of RBC death can potentially serve as a biomarker of the efficacy of vascular targeted cancer therapies.

Insights into photoacoustic speckle and applications in tumor characterization.

In ultrasound imaging, fully-developed speckle arises from the spatiotemporal superposition of pressure waves backscattered by randomly distributed scatterers. Speckle appearance is affected by the imaging system characteristics (lateral and axial resolution) and the random-like nature of the underlying tissue structure. In this work, we examine speckle formation in acoustic-resolution photoacoustic (PA) imaging using simulations and experiments. Numerical and physical phantoms were constructed to demonstrate that PA speckle carries information related to unresolved absorber structure in a manner similar to ultrasound speckle and unresolved scattering structures. A fractal-based model of the tumor vasculature was used to study PA speckle from unresolved cylindrical vessels. We show that speckle characteristics and the frequency content of PA signals can be used to monitor changes in average vessel size, linked to tumor growth. Experimental validation on murine tumors demonstrates that PA speckle can be utilized to characterize the unresolved vasculature in acoustic-resolution photoacoustic imaging.

Sizing biological cells using a microfluidic acoustic flow cytometer.

We describe a new technique that combines ultrasound and microfluidics to rapidly size and count cells in a high-throughput and label-free fashion. Using 3D hydrodynamic flow focusing, cells are streamed single file through an ultrasound beam where ultrasound scattering events from each individual cell are acquired. The ultrasound operates at a center frequency of 375 MHz with a wavelength of 4 µm; when the ultrasound wavelength is similar to the size of a scatterer, the power spectra of the backscattered ultrasound waves have distinct features at specific frequencies that are directly related to the cell size. Our approach determines cell sizes through a comparison of these distinct spectral features with established theoretical models. We perform an analysis of two types of cells: acute myeloid leukemia cells, where 2,390 measurements resulted in a mean size of 10.0 ± 1.7 µm, and HT29 colorectal cancer cells, where 1,955 measurements resulted in a mean size of 15.0 ± 2.3 µm. These results and histogram distributions agree very well with those measured from a Coulter Counter Multisizer 4. Our technique is the first to combine ultrasound and microfluidics to determine the cell size with the potential for multi-parameter cellular characterization using fluorescence, light scattering and quantitative photoacoustic techniques.

Simultaneous acoustic and photoacoustic microfluidic flow cytometry for label-free analysis.

We developed a label-free microfluidic acoustic flow cytometer (AFC) based on interleaved detection of ultrasound backscatter and photoacoustic waves from individual cells and particles flowing through a microfluidic channel. The AFC uses ultra-high frequency ultrasound, which has a center frequency of 375 MHz, corresponding to a wavelength of 4 µm, and a nanosecondpulsed laser, to detect individual cells. We validate the AFC by using it to count different color polystyrene microparticles and comparing the results to data from fluorescence-activated cell sorting (FACS). We also identify and count red and white blood cells in a blood sample using the AFC, and observe an excellent agreement with results obtained from FACS. This new label-free, non-destructive technique enables rapid and multi-parametric studies of individual cells of a large heterogeneous population using parameters such as ultrasound backscatter, optical absorption, and physical properties, for cell counting and sizing in biomedical and diagnostics applications.

Triplex micron-resolution acoustic, photoacoustic, and optical transmission microscopy via photoacoustic radiometry.

We present a new sensing technique, termed photoacoustic radiometry (PAR), for mapping the optical attenuation properties of a sample. In PAR, laser pulses attenuated via transmission through the sample impinge on the ultrasound transducer and generate a photoacoustic (PA) signal within it. Spatial variation of the optical attenuation properties of the sample influences the amplitude of the PAR signal, providing image contrast. Performed simultaneously with pulse-echo ultrasound and PA imaging, this triplex imaging technique enables rapid characterization of samples with micrometer-resolution in a single scan. In this work, we demonstrate that the PAR technique can be easily integrated into existing PA microscopy systems, with applications in imaging biological samples and non-destructive evaluation of optically opaque materials such as silicon wafers.

A Laser-Activated Biocompatible Theranostic Nanoagent for Targeted Multimodal Imaging and Photothermal Therapy.

Multifunctional nanoparticles have been reported for cancer detection and treatment currently. However, the accurate diagnosis and efficient treatment for tumors are still not satisfied. Here we report on the development of targeted phase change multimodal polymeric nanoparticles for the imaging and treatment of HER2-positive breast cancer. METHODS: We evaluated the multimodal imaging capabilities of the prepared nanoparticles in vitro using agar-based phantoms. The targeting performance and cytotoxicity of the nanoparticles were examined in cell culture using SKBR3 (over-expressing HER2) and MDA-MB-231 (HER2 negative) cells. We then tested the magnetic resonance (MR)/ photoacoustic (PA)/ ultrasound (US)/ near-infrared fluorescence (NIRF) multimodal imaging properties and photothermal effect of the nanoparticles in vivo using a SKBR3 breast xenograft model in nude mice. Tissue histopathology and immunofluorescence were also conducted. RESULTS: Both in vitro and in vivo systematical studies validated that the hybrid nanoparticles can be used as a superb MR/US/PA/NIRF contrast agent to simultaneously diagnose and guide tumor photothermal therapy (PTT). When irradiated by a near infrared laser, the liquid PFP vaporizes to a gas, rapidly expelling the contents and damaging surrounding tissues. The resulting micro-sized bubbles provide treatment validation through ultrasound imaging. Localization of DIR and SPIO in the tumor region facilitate photothermal therapy for targeted tumor destruction. The mice treated with HER2 targeted nanoparticles had a nearly complete response to treatment, while the controls showed continued tumor growth. CONCLUSION: This novel theranostic agent may provide better diagnostic imaging and therapeutic potential than current methods for treating HER2-positive breast cancer.

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.

Low-power noncontact photoacoustic microscope for bioimaging applications.

An inexpensive noncontact photoacoustic (PA) imaging system using a low-power continuous wave laser and a kilohertz-range microphone has been developed. The system operates in both optical and PA imaging modes and is designed to be compatible with conventional optical microscopes. Aqueous coupling fluids are not required for the detection of the PA signals; air is used as the coupling medium. The main component of the PA system is a custom designed PA imaging sensor that consists of an air-filled sample chamber and a resonator chamber that isolates a standard kilohertz frequency microphone from the input laser. A sample to be examined is placed on the glass substrate inside the chamber. A laser focused to a small spot by a 40 × objective onto the substrate enables generation of PA signals from the sample. Raster scanning the laser over the sample with micrometer-sized steps enables high-resolution PA images to be generated. A lateral resolution of 1.37 ?? ? m was achieved in this proof of concept study, which can be further improved using a higher numerical aperture objective. The application of the system was investigated on a red blood cell, with a noise-equivalent detection sensitivity of 43,887 hemoglobin molecules ( 72.88 × 10 ? 21 ?? mol or 72.88 zeptomol). The minimum pressure detectable limit of the system was 19.1 ?? ? Pa . This inexpensive, compact noncontact PA sensor is easily integrated with existing commercial optical microscopes, enabling optical and PA imaging of the same sample. Applications include forensic measurements, blood coagulation tests, and monitoring the penetration of drugs into human membrane.

Biodegradable polymeric nanoparticles containing gold nanoparticles and Paclitaxel for cancer imaging and drug delivery using photoacoustic methods.

In this study, optical-triggered multifunctional theranostic agents for photoacoustic/fluorescent imaging and cancer therapy have been developed. This system consists of a perfluorohexane liquid and gold nanoparticles (GNPs) in the core, stabilized by a Poly (lactide-co-glycolic acid) (PLGA) polymer shell. When cancer cells containing PLGA-GNPs were exposed to laser pulses, cell viability decreased due to the vaporization of the particles in and around the cells. The particle chemo drug loading and delivery capacity was also investigated in vitro experiments. These particles show potential as photoacoustic imaging and therapy agents for future clinical translation in cancer therapy.

High resolution ultrasound and photoacoustic imaging of single cells.

High resolution ultrasound and photoacoustic images of stained neutrophils, lymphocytes and monocytes from a blood smear were acquired using a combined acoustic/photoacoustic microscope. Photoacoustic images were created using a pulsed 532 nm laser that was coupled to a single mode fiber to produce output wavelengths from 532 nm to 620 nm via stimulated Raman scattering. The excitation wavelength was selected using optical filters and focused onto the sample using a 20× objective. A 1000 MHz transducer was co-aligned with the laser spot and used for ultrasound and photoacoustic images, enabling micrometer resolution with both modalities. The different cell types could be easily identified due to variations in contrast within the acoustic and photoacoustic images. This technique provides a new way of probing leukocyte structure with potential applications towards detecting cellular abnormalities and diseased cells at the single cell level.

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.

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.

Classification of blood cells and tumor cells using label-free ultrasound and photoacoustics.

A label-free method that can identify cells in a blood sample using high frequency photoacoustic and ultrasound signals is demonstrated. When the wavelength of the ultrasound or photoacoustic wave is similar to the size of a single cell (frequencies of 100-500 MHz), unique periodic features occur within the ultrasound and photoacoustic power spectrum that depend on the cell size, structure, and morphology. These spectral features can be used to identify different cell types present in blood, such as red blood cells (RBCs), white blood cells (WBCs), and circulating tumor cells. Circulating melanoma cells are ideal for photoacoustic detection due to their endogenous optical absorption properties. Using a 532 nm pulsed laser and a 375 MHz transducer, the ultrasound and photoacoustic signals from RBCs, WBCs, and melanoma cells were individually measured in an acoustic microscope to examine how the signals change between cell types. A photoacoustic and ultrasound signal was detected from RBCs and melanoma cells; only an ultrasound signal was detected from WBCs. The different cell types were distinctly separated using the ultrasound and photoacoustic signal amplitude and power spectral periodicity. The size of each cell was also estimated from the spectral periodicity. For the first time, sound waves generated using pulse-echo ultrasound and photoacoustics have been used to identify and size single cells, with applications toward counting and identifying cells, including circulating melanoma cells.

Modeling photoacoustic spectral features of micron-sized particles.

The photoacoustic signal generated from particles when irradiated by light is determined by attributes of the particle such as the size, speed of sound, morphology and the optical absorption coefficient. Unique features such as periodically varying minima and maxima are observed throughout the photoacoustic signal power spectrum, where the periodicity depends on these physical attributes. The frequency content of the photoacoustic signals can be used to obtain the physical attributes of unknown particles by comparison to analytical solutions of homogeneous symmetric geometric structures, such as spheres. However, analytical solutions do not exist for irregularly shaped particles, inhomogeneous particles or particles near structures. A finite element model (FEM) was used to simulate photoacoustic wave propagation from four different particle configurations: a homogeneous particle suspended in water, a homogeneous particle on a reflecting boundary, an inhomogeneous particle with an absorbing shell and non-absorbing core, and an irregularly shaped particle such as a red blood cell. Biocompatible perfluorocarbon droplets, 3-5 µm in diameter containing optically absorbing nanoparticles were used as the representative ideal particles, as they are spherical, homogeneous, optically translucent, and have known physical properties. The photoacoustic spectrum of micron-sized single droplets in suspension and on a reflecting boundary were measured over the frequency range of 100-500 MHz and compared directly to analytical models and the FEM. Good agreement between the analytical model, FEM and measured values were observed for a droplet in suspension, where the spectral minima agreed to within a 3.3 MHz standard deviation. For a droplet on a reflecting boundary, spectral features were correctly reproduced using the FEM but not the analytical model. The photoacoustic spectra from other common particle configurations such as particle with an absorbing shell and a biconcave-shaped red blood cell were also investigated, where unique features in the power spectrum could be used to identify them.

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.