Controlled Tempering of Lipid Concentration and Microbubble Shrinkage as a Possible Mechanism for Fine-Tuning Microbubble Size and Shell Properties.

The acoustic response of microbubbles (MBs) depends on their resonance frequency, which is dependent on the MB size and shell properties. Monodisperse MBs with tunable shell properties are thus desirable for optimizing and controlling the MB behavior in acoustics applications. By utilizing a novel microfluidic method that uses lipid concentration to control MB shrinkage, we generated monodisperse MBs of four different initial diameters at three lipid concentrations (5.6, 10.0, and 16.0 mg/mL) in the aqueous phase. Following shrinkage, we measured the MB resonance frequency and determined its shell stiffness and viscosity. The study demonstrates that we can generate monodisperse MBs of specific sizes and tunable shell properties by controlling the MB initial diameter and aqueous phase lipid concentration. Our results indicate that the resonance frequency increases by 180-210% with increasing lipid concentration (from 5.6 to 16.0 mg/mL), while the bubble diameter is kept constant. Additionally, we find that the resonance frequency decreases by 260-300% with an increasing MB final diameter (from 5 to 12 µm), while the lipid concentration is held constant. For example, our results depict that the resonance frequency increases by ∼195% with increasing lipid concentration from 5.6 to 16.0 mg/mL, for ∼11 µm final diameter MBs. Additionally, we find that the resonance frequency decreases by ∼275% with increasing MB final diameter from 5 to 12 µm when we use a lipid concentration of 5.6 mg/mL. We also determine that MB shell viscosity and stiffness increase with increasing lipid concentration and MB final diameter, and the level of change depends on the degree of shrinkage experienced by the MB. Specifically, we find that by increasing the concentration of lipids from 5.6 to 16.0 mg/mL, the shell stiffness and viscosity of ∼11 µm final diameter MBs increase by ∼400 and ∼200%, respectively. This study demonstrates the feasibility of fine-tuning the MB acoustic response to ultrasound by tailoring the MB initial diameter and lipid concentration.

Microfluidic nanobubbles: observations of a sudden contraction of microbubbles into nanobubbles.

Microfluidic devices are often utilized to generate uniform-size microbubbles. In most microfluidic bubble generation experiments, once the bubbles are formed the gas inside the bubbles begin to dissolve into the surrounding aqueous environment. The bubbles shrink until they attain an equilibrium size dictated by the concentration and type of amphiphilic molecules stabilizing the gas-liquid interface. Here, we exploit this shrinkage mechanism, and control the solution lipid concentration and microfluidic geometry, to make monodisperse bulk nanobubbles. Interestingly, we make the surprising observation of a critical microbubble diameter above and below which the scale of bubble shrinkage dramatically changes. Namely, microbubbles generated with an initial diameter larger than the critical diameter shrinks to a stable diameter that is consistent with previous literature. However, microbubbles that are initially smaller than the critical diameter experience a sudden contraction into nanobubbles whose size is at least an order-of-magnitude below expectations. We apply electron microscopy and resonance mass measurement methods to quantify the size and uniformity of the nanobubbles, and probe the dependence of the critical bubble diameter on the lipid concentration. We anticipate that further analysis of this unexpected microbubble sudden contraction regime can lead to more robust technologies for making monodisperse nanobubbles.

Objective assessment of stored blood quality by deep learning.

Stored red blood cells (RBCs) are needed for life-saving blood transfusions, but they undergo continuous degradation. RBC storage lesions are often assessed by microscopic examination or biochemical and biophysical assays, which are complex, time-consuming, and destructive to fragile cells. Here we demonstrate the use of label-free imaging flow cytometry and deep learning to characterize RBC lesions. Using brightfield images, a trained neural network achieved 76.7% agreement with experts in classifying seven clinically relevant RBC morphologies associated with storage lesions, comparable to 82.5% agreement between different experts. Given that human observation and classification may not optimally discern RBC quality, we went further and eliminated subjective human annotation in the training step by training a weakly supervised neural network using only storage duration times. The feature space extracted by this network revealed a chronological progression of morphological changes that better predicted blood quality, as measured by physiological hemolytic assay readouts, than the conventional expert-assessed morphology classification system. With further training and clinical testing across multiple sites, protocols, and instruments, deep learning and label-free imaging flow cytometry might be used to routinely and objectively assess RBC storage lesions. This would automate a complex protocol, minimize laboratory sample handling and preparation, and reduce the impact of procedural errors and discrepancies between facilities and blood donors. The chronology-based machine-learning approach may also improve upon humans' assessment of morphological changes in other biomedically important progressions, such as differentiation and metastasis.

Controlled Shrinkage of Microfluidically Generated Microbubbles by Tuning Lipid Concentration.

Monodisperse microbubbles with diameters less than 10 µm are desirable in several ultrasound imaging and therapeutic delivery applications. However, conventional approaches to synthesize microbubbles, which are usually agitation-based, produce polydisperse bubbles that are less desirable because of their heterogeneous response when exposed to an ultrasound field. Microfluidics technology has the unique advantage of generating size-controlled monodisperse microbubbles, and it is now well established that the diameter of microfluidically made microbubbles can be tuned by varying the liquid flow rate, gas pressure, and dimensions of the microfluidic channel. It is also observed that once the microbubbles form, the bubbles shrink and eventually stabilize to a quasi-equilibrium diameter, and that the rate of stabilization is related to the lipid solution. However, how the lipid solution concentration affects the degree of bubble shrinkage, and the stable size of microbubbles, has not been thoroughly examined. Here, we investigate whether and how the lipid concentration affects the degree of microbubble shrinkage. Namely, we utilize a flow-focusing microfluidic geometry to generate monodisperse bubbles, and observe the effect of gas composition (2.5, 1.42, and 0.17 wt % octafluoropropane in nitrogen) and lipid concentration (1-16 mg/mL) on the degree of microbubble shrinkage. For the lipid system and gas utilized in these experiments, we observe a monotonic increase in the degree of microbubble shrinkage with decreasing lipid concentration, and no dependency on the gas composition. We hypothesize that the degree of shrinkage is related to lipid concentration by the self-assembly of lipids on the gas-liquid interface during bubble generation and subsequent lipid packing on the interface during shrinkage, which is arrested when a maximum packing density is achieved. We anticipate that this approach for creating and tuning the size of monodisperse microbubbles will find utility in biomedical applications, such as contrast-enhanced ultrasound imaging and ultrasound-triggered gene delivery.

Microfluidic Generation of Monodisperse Nanobubbles by Selective Gas Dissolution.

Nanotechnology currently enables the fabrication of uniform solid nanoparticles and liquid nano-emulsions, but not uniform gaseous nanobubbles (NBs). In this article, for the first time, a method based on microfluidics that directly produces monodisperse NBs is reported. Specifically, a two-component gas mixture of water-soluble nitrogen and water-insoluble octafluoropropane as the gas phase are used in a microfluidic bubble generator. First, monodisperse microbubbles (MBs) with a classical microfluidic flow-focusing junction is generated, then the MBs shrink down to ≈100 nm diameter, due to the dissolution of the water-soluble components in the gas mixture. The degree of shrinkage is controlled by tuning the ratio of water-soluble to water-insoluble gas components. This technique maintains the monodispersity of the NBs, and enables precise control of the final NB size. It is found that the monodisperse NBs show better homogeneity than polydisperse NBs in in vitro ultrasound imaging experiments. Proof-of-concept in vivo kidney imaging is performed in live mice, demonstrating enhanced contrast using the monodisperse NBs. The NB monodispersity and imaging results make microfluidically generated NBs promising candidates as ultrasound contrast and molecular imaging agents.

Bursting Microbubbles: How Nanobubble Contrast Agents Can Enable the Future of Medical Ultrasound Molecular Imaging and Image-Guided Therapy.

The field of medical ultrasound has undergone a significant evolution since the development of microbubbles as contrast agents. However, due to their size, microbubbles remain in the vasculature, and therefore have limited clinical applications. Building a better - and smaller - bubble can expand the applications of contrast-enhanced ultrasound by allowing bubbles to extravasate from blood vessels - creating new opportunities. In this review, we summarize recent research on the formulation and use of NBs as imaging agents and as therapeutic vehicles. We discuss the ongoing debates in the field and reluctance to accepting NBs as an acoustically active construct and a potentially impactful clinical tool that can help shape the future of medical ultrasound. We hope that the overview of key experimental and theoretical findings in the NB field presented in this paper provides a fundamental framework that will help clarify NB-ultrasound interactions and inspire engagement in the field.

Dancing with the Cells: Acoustic Microflows Generated by Oscillating Cells.

The interaction of a sound or ultrasound wave with an elastic object, such as a microbubble, can give rise to a steady-state microstreaming flow in its surrounding liquid. Many microfluidic strategies for cell and particle manipulation, and analyte mixing, are based on this type of flow. In addition, there are reports that acoustic streaming can be generated in biological systems, for instance, in a mammalian inner ear. Here, new observations are reported that individual cells are able to induce microstreaming flow, when they are excited by controlled acoustic waves in vitro. Single adherent cells are exposed to an acoustic field inside a microfluidic device. The cell-induced microstreaming is then investigated by monitoring flow tracers around the cell, while the structure and extracellular environment of the cell are altered using different chemicals. The observations suggest that the maximum streaming flow induced by an MDA-MB-231 breast cancer cell can reach velocities on the order of mm s-1 , and this maximum velocity is primarily governed by the overall cell stiffness. Therefore, such cell-induced microstreaming measurements, including flow pattern and velocity magnitude, may be used as label-free proxies of cellular mechanical properties, such as stiffness.

Emerging use of machine learning and advanced technologies to assess red cell quality.

Improving blood product quality and patient outcomes is an accepted goal in transfusion medicine research. Thus, there is an urgent need to understand the potential adverse effects on red blood cells (RBCs) during pre-transfusion storage. Current assessment techniques of these degradation events, termed "storage lesions", are subjective, labor-intensive, and complex. Here we describe emerging technologies that assess the biochemical, biophysical, and morphological characteristics of RBC storage lesions. Of these emerging techniques, machine learning (ML) has shown potential to overcome the limitations of conventional RBC assessment methods. Our previous work has shown that neural networks can extract chronological progressions of morphological changes in RBCs during storage without human input. We hypothesize that, with broader training and testing of multivariate data (e.g., varying donor factors and manufacturing methods), ML can further our understanding of clinical transfusion outcomes in multiple patient groups.

Label-Free Analysis of Red Blood Cell Storage Lesions Using Imaging Flow Cytometry.

Deleterious changes, collectively termed as storage lesions, alter the characteristics of red blood cell (RBC) morphology during in vitro storage. Due to gradual loss of cellular membrane, RBCs lose their original biconcave disk shape and begin a process of spherical deformation that is characterized by well-defined morphological criteria. At the spheroechinocyte stage, the structure of RBC is irreversibly damaged and lacks the elasticity necessary to efficiently deliver oxygen. Quantifying the prevalence of spheroechinocytes could provide an important morphological measure of the quality of stored blood products. Unlike the conventional RBC morphology characterization assay involving light microscopy, we introduce a label-free assay using imaging flow cytometry (IFC). The technique captures 100,000 images of a sample and calculates a relative measure of spheroechinocyte population in a fraction of the time required by the conventional method. A comparative method study, measuring a morphological index for 11 RCC units through storage, found that the two techniques measured similar trends with IFC reporting the metric at an average of 3.9% higher. We monitored 18 RCC units between Weeks 1 and 6 of storage and found that the spheroechinocyte population increased by an average of 26.2%. The large (3.5-64.1%) variation between the units' spheroechinocyte population percentage at Week 1 suggests a possible dependence of blood product quality on donor characteristics. Given our method's ability to rapidly monitor large samples and refine morphological characterization beyond conventional methods, we believe our technique offers good potential for studying the underlying relationships between RBC morphology and blood storage lesions. © 2019 International Society for Advancement of Cytometry.

Feasibility of photoacoustic imaging for the non-invasive quality management of stored blood bags.

BACKGROUND AND OBJECTIVES: During the in vitro storage of red blood cells (RBCs), unfavourable changes (storage lesions) cause a rapid consumption of intracellular diphosphoglycerate. The latter deregulates the oxygen-haemoglobin binding potential, subsequently increasing oxygen saturation (SO2 ) and membrane degradation, transforming RBCs from biconcave discs to rigid spherical bodies (spheroechinocytes). Current laboratory techniques invasively extract RBC samples to assess the quality of red cell concentrate (RCC) units. Optical technologies could provide a means of assessing quality non-invasively. MATERIALS AND METHODS: A photoacoustic (PA) imaging technique was developed for acquiring the SO2 of blood bags non-invasively. Seven RCC units were monitored every 3-5 days until expiry (6 weeks). Measurements were validated against a conventional blood gas analyzer (BGA). Using an image flow cytometry assay, morphological profile trends were compared against the SO2 trends during blood bag storage. RESULTS: A strong correlation (r2  ≥ 0·95) was found when comparing temporal data between PA and BGA SO2 measurements. Inter-sample PA variability was found to be similar to that produced by BGA (±0·8%). A strong correlation was found to exist between the temporal changes in SO2 and relative spheroechinocyte population (0·79 ≤ r2  ≤ 0·97). CONCLUSION: This study suggests that PA imaging can non-invasively track the SO2 of stored RBCs non-invasively. By longitudinally monitoring the change in SO2 , it is possible to infer the effects of the storage lesion on RBC morphology. This non-invasive monitoring technique allows for the assessment of blood bags, without compromising sterility pre-transfusion.

Feasibility of detecting change in backscattered energy of acoustic harmonics in locally heated tissues.

Purpose: A real-time noninvasive thermometry technique is required to estimate the temperature distribution during hyperthermia to monitor and control the treatment. The main objective of this study is to demonstrate the possibility of detecting change in backscatter energy (CBE) of acoustic harmonics in tissue-mimicking gel phantoms and ex vivo bovine muscle tissues in which the temperature was locally increased within the hyperthermia regime. Materials and Methods: A peristaltic pump was used to circulate hot water through a needle inserted inside the samples to locally increase the temperature from 26 °C to 46 °C. The CBE of acoustic harmonics were used to identify the location of temperature changes in the samples. A conventional echo-shift technique was also implemented for comparison. Data collection was performed for two conditions to investigate the effect of motion on both techniques by: (1) inducing vibration in the sample through the peristatic pump and, (2) subsequently with no sample vibration while the pump was off. Results: Harmonics were able to determine the location of temperature rise in the presence and absence of vibration. In gel phantom, the mean contrast to noise ratio (CNR) in CBE maps reduced by a factor of 0.86 due to vibration whereas in gradient maps the CNR reduced by a factor of 8.3. Conclusions: The findings of this study suggest that the change in backscatter energy of acoustic harmonics can potentially be used to develop a noninvasive ultrasound-based thermometry technique with lower susceptibility to motion artifacts compared to the echo-shift method.

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.

Intrinsically absorbing photoacoustic and ultrasound contrast agents for cancer therapy and imaging.

Nanoparticles are submicrometer in size and are used in a variety of ways in the biomedical field. They can carry therapeutic drugs, either in the particle core or surface to target cancer sites in the body. Additionally they can contain imaging agents to diagnose and monitor the tumor size using different imaging modalities, such as fluorescence and nuclear magnetic resonance imaging. Novel theranostic nanoparticle agents, called perfluorohexane nanoemulsions (PFH-NEs) were synthesized whose intrinsic properties could be used for both imaging (ultrasound and photoacoustic) and therapy. Compared to other theranostic agents, our PFH-NEs can absorb sufficient near-infrared light to enhance contrast and provide deeper penetration imaging at laser fluences causing minimal damage to healthy tissue. One contrast mechanism (optical absorption/photoacoustics) allows us to validate localization of the agent and another (acoustic impedance/ultrasound) allows the imaging of therapeutic delivery after particle activation. In this work, we show the potential of these PFH-NEs to be used as multimodal imaging agents and for therapy.

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.

Honey, I shrunk the bubbles: microfluidic vacuum shrinkage of lipid-stabilized microbubbles.

We present a microfluidic technique that shrinks lipid-stabilized microbubbles from O(100) to O(1) µm in diameter - the size that is desirable in applications as ultrasound contrast agents. We achieve microbubble shrinkage by utilizing vacuum channels that are adjacent to the microfluidic flow channels to extract air from the microbubbles. We tune a single parameter, the vacuum pressure, to accurately control the final microbubble size. Finally, we demonstrate that the resulting O(1) µm diameter microbubbles have similar stability to microfluidically generated microbubbles that are not exposed to vacuum shrinkage. We anticipate that, with additional scale-up, this simple approach to shrink microbubbles generated microfluidically will be desirable in ultrasound imaging and therapeutic applications.

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.

Synthesis of Stable Multifunctional Perfluorocarbon Nanoemulsions for Cancer Therapy and Imaging.

Nanotechnology provides a promising platform for drug-delivery in medicine. Nanostructured materials can be designed with desired superparamagnetic or fluorescent properties in conjunction with biochemically functionalized moieties (i.e., antibodies, peptides, and small molecules) to actively bind to target sites. These multifunctional properties make them suitable agents for multimodal imaging, diagnosis, and therapy. Perfluorohexane nanoemulsions (PFH-NEs) are novel drug-delivery vehicles and contrast agents for ultrasound and photoacoustic imaging of cancer in vivo, offering higher spatial resolution and deeper penetration of tissue when compared to conventional optical techniques. Compared to other theranostic agents, our PFH-NEs are one of the smallest of their kind (<100 nm), exhibit minimal aggregation, long-term stability at physiological conditions, and provide a noninvasive cancer imaging and therapy alternative for patients. Here, we show, using high-resolution imaging and correlative techniques, that our PFH-NEs, when in tandem with silica-coated gold nanoparticles (scAuNPs), can be used as a drug-loaded therapeutic via endocytosis and as a multimodal imaging agent for photoacoustic, ultrasound, and fluorescence imaging of tumor growth.

Temperature dependence of acoustic harmonics generated by nonlinear ultrasound wave propagation in water at various frequencies.

Ultrasound-based thermometry requires a temperature-sensitive acoustic parameter that can be used to estimate the temperature by tracking changes in that parameter during heating. The objective of this study is to investigate the temperature dependence of acoustic harmonics generated by nonlinear ultrasound wave propagation in water at various pulse transmit frequencies from 1 to 20 MHz. Simulations were conducted using an expanded form of the Khokhlov-Zabolotskaya-Kuznetsov nonlinear acoustic wave propagation model in which temperature dependence of the medium parameters was included. Measurements were performed using single-element transducers at two different transmit frequencies of 3.3 and 13 MHz which are within the range of frequencies simulated. The acoustic pressure signals were measured by a calibrated needle hydrophone along the axes of the transducers. The water temperature was uniformly increased from 26 °C to 46 °C in increments of 5 °C. The results show that the temperature dependence of the harmonic generation is different at various frequencies which is due to the interplay between the mechanisms of absorption, nonlinearity, and focusing gain. At the transmit frequencies of 1 and 3.3 MHz, the harmonic amplitudes decrease with increasing the temperature, while the opposite temperature dependence is observed at 13 and 20 MHz.

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.