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We present a microfluidic technique that shrinks lipidstabilized 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 microfluidics 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 therapeutics applications.

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.

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 (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.

In this work, we explored the potential of measuring shear wave propagation using optical coherence elastography (OCE) in an inhomogeneous phantom and carotid artery samples based on a sweptsource optical coherence tomography (OCT) system. Shear waves were generated using a piezoelectric transducer transmitting sine-wave bursts of 400 μs duration, applying acoustic radiation force (ARF) to inhomogeneous phantoms and carotid artery samples, synchronized with a swept-source OCT (SS-OCT) imaging system. The phantoms were composed of gelatin and titanium dioxide whereas the carotid artery samples were embedded in gel. Differential OCT phase maps, measured with and without the ARF, detected the microscopic displacement generated by shear wave propagation in these phantoms and samples of different stiffness. We present the technique for calculating tissue mechanical properties by propagating shear waves in inhomogeneous tissue equivalent phantoms and carotid artery samples using the ARF of an ultrasound transducer, and measuring the shear wave speed and its associated properties in the different layers with OCT phase maps. This method lays the foundation for future in-vitro and in-vivo studies of mechanical property measurements of biological tissues such as vascular tissues, where normal and pathological structures may exhibit significant contrast in the shear modulus.

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 PLGAGNPs 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.

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.

Perfluorocarbon droplets containing nanoparticles (NPs) have recently been investigated as theranostic and dual-mode contrast agents. These droplets can be vaporized via laser irradiation or used as photoacoustic contrast agents below the vaporization threshold. This study investigates the photoacoustic mechanism of NP-loaded droplets using photoacoustic frequencies between 100 and 1000 MHz, where distinct spectral features are observed that are related to the droplet composition. The measured photoacoustic spectrum from NP-loaded perfluorocarbon droplets was compared to a theoretical model that assumes a homogenous liquid. Good agreement in the location of the spectral features was observed, which suggests the NPs act primarily as optical absorbers to induce thermal expansion of the droplet as a single homogenous object. The NP size and composition do not affect the photoacoustic spectrum; therefore, the photoacoustic signal can be maximized by optimizing the NP optical absorbing properties. To confirm the theoretical parameters in the model, photoacoustic, ultrasonic, and optical methods were used to estimate the droplet diameter. Photoacoustic and ultrasonic methods agreed to within 1.4%, while the optical measurement was 8.5% higher; this difference decreased with increasing droplet size. The small discrepancy may be attributed to the difficulty in observing the small droplets through the partially translucent phantom.

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. Keywords: Photoacoustic speckle, Ultrasound speckle, Spatial resolution, Autocovarience function, Tumor vasculature, In-vivo imaging, Vascular trees

Objective: This work aims to determine whether photoacoustic (PA) thermometry from a commercially available PA imaging system can be used to control the temperature in nanoparticle-mediated thermal therapies. Methods: The PA imaging system was interfaced to obtain PA images while scanning ex-vivo tissue. These images were then used to obtain temperature maps in real-time during heating. Validation and calibration of the PA thermometry were done using a fluoroptic thermometer. This thermometer was also used to develop and tune a software-based proportional integral derivative (PID) controller. Finally, a PA-based PID closed-loop controller was used to control gold nanorod (GNR) mediated laser therapy. Results: The use of GNRs substantially enhanced laser heating; the temperature rise increased 7-fold by injecting a GNR solution with a concentration of 0.029 mg/mL. The control experiments showed that the desired temperature could be achieved and maintained at a targeted location in the ex-vivo tissue. The steady-state mean absolute deviations (MAD) from the targeted temperature during control were between 0.16 $^\circ {\kern-0.70007pt}\text{C}$ and 0.5 $^\circ {\kern-0.70007pt}\text{C}$ , depending on the experiment. Conclusion: It was possible to control hyperthermia treatments using a software-based PID controller and a commercial PA imaging system. Significance: The monitoring and control of the temperature in thermal-based therapies are important for assuring a prescribed temperature to the target tissue while minimizing the temperature of the surrounding healthy tissue. This easily implemented non-invasive control system will facilitate the realization of a broad range of hyperthermia treatments.

Optoacoustic imaging is a relatively novel biomedical imaging modality that relies on the absorption of light to create pressure transients that can be detected ultrasonically. In most scientific communications, the source of tissue contrast has been described as primarily optical. However, the thermomecahnical properties of tissue, as expressed through the Gruneisen coefficient, also affect the optoacoustic signal. To investigate the effect of thermomechanical tissue properties short pulses (~ 6.5 ns) from an optical parametric oscillator at 750 nm were used to irradiate coagulated and uncoagulated tissue-mimicking albumen phantoms, to emulate normal tissue and tissue that has been heated. The phantoms respond to the laser-induced stress by thermoelastic expansion. This thermomechanical behavior of the samples was assessed using an interferometric system capable of measuring transient displacements with a temporal resolution of less than 10 ns and a spatial resolution of < 10 nm. The experimental measurement allowed determination of the Gruneisen coefficient which is an important thermo-mechanical sample property that can affect generation of optoacoustic signals. An increase in the value of Gruneisen coefficient of 65% was measured when phantoms were coagulated compared to uncoagulated phantoms, consistent with the stiffening of the tissue mimicking material. This suggests that for thermal therapy the changes in the Gruneisen coefficient are also an important source of optoacoustic contrast.

Optoacoustic Imaging is based on the thermal expansion of tissue caused by a temperature rise due to absorption of short laser pulses. At constant laser fluence, optoacoustic image contrast is proportional to differences in optical absorption and the thermoacoustic efficiency, expressed by the Grneisen parameter, Γ. Γ is proportional to the thermal expansion coefficient, the sound velocity squared and the inverse heat capacity at constant pressure. In thermal therapies, these parameters may be modified in the treated area. In this work experiments were performed to examine the influence of these parameters on image contrast. A Laser Optoacoustic Imaging System (LOIS, Fairway Medical Technologies, Houston, Texas) was used to image tissue phantoms comprised of cylindrical Polyvinyl Chloride Plastisol (PVCP) optical absorbing targets imbedded in either gelatin or PVCP as the background medium. Varying concentrations of Black Plastic Color (BPC) and titanium dioxide (TiO2)were added to targets and background to yield desired tissue relevant optical absorption and effective scattering coefficients, respectively. In thermal therapy experiments, ex-vivo bovine liver was heated with laser fibres (805nm laser at 5 W for 600s) to create regions of tissue coagulation. Lesions formed in the liver tissue were visible using the LOIS system with reasonable correspondence to the actual region of tissue coagulation. In the phantom experiments, contrast could be seen with low optical absorbing targets (μaof 0.50cm−1down to0.13cm−1)embedded in a gelatin background (μa=0.13cm−1andμs′=4.2cm−1). Therefore, the data suggest that small objects (

Understanding the pressure dependence of the nonlinear behavior of ultrasonically excited phospholipid-stabilized nanobubbles (NBs) is important for optimizing ultrasound exposure parameters for implementations of contrast enhanced ultrasound, critical to molecular imaging. The viscoelastic properties of the shell can be controlled by the introduction of membrane additives, such as propylene glycol as a membrane softener or glycerol as a membrane stiffener. We report on the production of high-yield NBs with narrow dispersity and different shell properties. Through precise control over size and shell structure, we show how these shell components interact with the phospholipid membrane, change their structure, affect their viscoelastic properties, and consequently change their acoustic response. A two-photon microscopy technique through a polarity-sensitive fluorescent dye, C-laurdan, was utilized to gain insights on the effect of membrane additives to the membrane structure. We report how the shell stiffness of NBs affects the pressure threshold (Pt) for the sudden amplification in the scattered acoustic signal from NBs. For narrow size NBs with 200 nm mean size, we find Pt to be between 123 and 245 kPa for the NBs with the most flexible membrane as assessed using C-Laurdan, 465-588 kPa for the NBs with intermediate stiffness, and 588-710 kPa for the NBs with stiff membranes. Numerical simulations of the NB dynamics are in good agreement with the experimental observations, confirming the dependence of acoustic response to shell properties, thereby substantiating further the development in engineering the shell of ultrasound contrast agents. The viscoelastic-dependent threshold behavior can be utilized for significantly and selectively enhancing the diagnostic and therapeutic ultrasound applications of potent narrow size NBs.

Microbubbles (MBs) stabilized by particle surfactants (i.e., Pickering bubbles) have better thermodynamic stability compared to MBs stabilized by small molecules as a result of steric hindrance against coalescence, higher diffusion resistance, and higher particle desorption energy. In addition, the use of particles to stabilize MBs that are typically used as an ultrasound (US) contrast agent can also introduce photoacoustic (PA) properties, thus enabling a highly effective dual-modality US and PA contrast agent. Here, we report the use of partially reduced and functionalized graphene oxide as the sole surfactant to stabilize perfluorocarbon gas bubbles in the preparation of a dual-modality US and PA agent, with high contrast in both imaging modes and without the need for small-molecule or polymer additives. This approach offers an increase in loading of the PA agent without destabilization and increased thickness of the MB shell compared to traditional systems, in which the focus is on adding a PA agent to existing MB formulations.

A real-time and non-invasive thermometry method is crucial for thermal therapies to monitor and control the treatment. Ultrasound can be used as an attractive thermometry modality for its relatively high sensitivity to change in temperature and fast data collection and processing abilities. In this work, an ultrasound thermometry method based on the change in backscattered energy (CBE) is used to control the tissue temperature using a closed-loop controller in real-time. A clinical high-frequency ultrasound scanner was used to acquire RF echo data from ex vivo porcine tissue samples while the tissue was being exposed to an interstitial laser heating source. The control system was used to rapidly increase the temperature from 37°C (baseline temperature) to 43 °C (target temperature) and maintain the target temperature for about 6 minutes. The results show that the ultrasound thermometry based on CBE generated by a high-frequency ultrasound scanner can be used to generate 2D temperature maps of a localized heating region in the hyperthermia temperature range (∼43°C). The estimated temperature varied by an average of ±0.8 °C compared to a calibrated fiber-optic measurement. Thus, a non-invasive ultrasound thermometry method based on the CBE technique can be used for real-time monitoring and control of hyperthermia treatments, using the interstitial laser heating source with acceptable accuracy.

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.

Background: Minimally invasive modalities are of great interest in the field of treating bone tumors. However, providing reliable mechanical support and fast killing of tumor cells to achieve rapid recovery of physical function is still challenging in clinical works. Methods: A material with two functions, mechanical support and magnetic thermal ablation, was developed from Fe3O4 nanoparticles (NPs) distributed in a polymethylmethacrylate (PMMA) bone cement. The mechanical properties and efficiency of magnetic field-induced thermal ablation were systematically and successfully evaluated in vitro and ex vivo. CT images and pathological examination were successfully applied to evaluate therapeutic efficacy with a rabbit bone tumor model. Biosafety evaluation was performed with a rabbit in vivo, and a cytotoxicity test was performed in vitro. Results: An NP content of 6% Fe3O4 (PMMA-6% Fe3O4, mFe: 0.01 g) gave the most suitable performance for in vivo study. At the 56-day follow-up after treatment, bone tumors were ablated without obvious side effects. The pathological examination and new bone formation in CT images clearly illustrate that the bone tumors were completely eliminated. Correspondingly, after treatment, the tendency of bone tumors toward metastasis significantly decreased. Moreover, with well-designed mechanical properties, PMMA-6%Fe3O4 implantation endowed tumor-bearing rabbit legs with excellent bio-mimic bone structure and internal support. Biosafety evaluation did not induce an increase or decrease in the immune response, and major functional parameters were all at normal levels. Conclusion: We have presented a novel, highly efficient and minimally invasive approach for complete bone tumor regression and bone defect repair by magnetic thermal ablation based on PMMA containing Fe3O4 NPs; this approach shows excellent heating ability for rabbit VX2 tibial plateau tumor ablation upon exposure to an alternating magnetic field (AMF) and provides mechanical support for bone repair. The new and powerful dual-function implant is a promising minimally invasive agent for the treatment of bone tumors and has good clinical translation potential.

It has been previously shown that high frequency ultrasound (20 - 100 MHz) can be used to detect cellular structure changes in tissues and cell ensembles. Using spectral analysis methods to analyze radio-frequency data collected from in vitro and in vivo models, the changes seen during apoptotic cell death are very striking. Imaging changes in cell structure has implications in a broad range of fields, from cancer treatment monitoring to organ transplantation. However, the changes seen in the backscattered ultrasound intensity and frequency spectrum are not fully understood. In this paper we propose and explore a model for studying how the changes in the sizes, spatial distribution, and acoustic impedance of the scattering sources within the cells are related to the resulting backscattered ultrasound signal

The goal of this project is to investigate changes in the acoustical properties of cells undergoing cell death for the development of a method for tissue apoptosis detection using high frequency ultrasound (10-60 MHz). A scanning acoustic microscope (SAM) was used for visualization of individual cells undergoing apoptosis (SASAM, Fraunhofer IBMT, Germany). The use of the SAM offers high resolution (1 μm spot size) and therefore enables the exploration of acoustical properties of the cell nucleus. Cells were labeled with H33342 and DIOC 3(5) for visualizing condensed chromatin and membranes in fluorescence microscopy. In addition the same cell lines interrogated microscopically were investigated using high frequency ultrasound. Recorded radio frequency (rf) data were analyzed using ultrasound spectroscopy. Integrated backscatter coefficients and attenuation values were computed for two cell lines: HeLa and MDCK. Both cell lines responded to the applied chemotherapeutic agent by apoptosis, assessed by fluorescence microscopy. Acoustical and optical microscopy using the SASAM system clearly enabled a differentiation between apoptotic cells and cells not responding to the treatment. Apoptotic cells displayed a higher contrast in the acoustic images and were less regular in shape. Optical images of the same cells showed nuclear condensation and membrane disruption. Spectral parameters estimated from rf ultrasound showed a 100% increase in the integrated backscatter coefficients for HeLa and MDCK. Attenuation values were increased by 50% to 70% for both cell lines as a function of treatment time. The results of this investigation provide a better understanding of changes in the acoustical properties of cells with cell death and thus to the development of a non-invasive method for measuring the treatment response of tumors using acoustic waves.

A theoretical model examining the effects of erythrocyte oxygenation on optoacoustic (OA) signals is presented. Each erythrocyte is considered as a fluid sphere and its optical absorption is defined by its oxygen saturation state. The OA field generated by a cell is computed by solving the wave equation in the frequency domain with appropriate boundary conditions. The resultant field from many cells is simulated by summing the pressure waves emitted by individual cells. A Monte Carlo algorithm generates 2-D spatially random distributions of oxygenated and deoxygenated erythrocytes. Oxygen saturation levels of oxygenated cells a assumed to be 100% and 0% for deoxygenated cells. The OA signal amplitude decreases monotonically for the 700-nm laser source and increases monotonically for 1000 nm optical radiation when blood oxygen saturation varies from 0 to 100%. An approximately sixfold decrease and fivefold increase of the OA signal amplitude were computed at those wavelengths, respectively. The OA spectral power in the low-frequency range (100 MHz) decreases for 700 nm and increases for 1000 nm with increasing blood oxygen saturation. This model provides a theoretical framework to study the erythrocyte oxygenation-dependent OA signals.

In a previous experiment [Baddouret al., J. Acoust. Soc. Am.117(2), 934–943 (2005)] it was shown that it is possible to deduce the ultra-sound backscatter transfer function from single, subresolution cells in vitro, across a broad, continuous range of frequencies. Additional measurements have been performed at high frequencies (10 – 65 MHz) on cells with different relative nucleus sizes. It was found that for cells with a nucleus to cell volume ratio of 0.50, the backscatter response was better modeled as an elastic sphere. For the cells in which the ratio was 0.33, the backscatter showed good agreement with the theoretical solution for a fluid sphere.

Nanobubbles have gained attention for their use as highly stable ultrasound (US) contrast agents, but assessment of individual nanobubble size remains a challenge. Current sizing techniques require either extensive sample preparation or depend on assumed values of nanobubble density that are not well characterized. An US based approach would be desirable; however, probing individual nanobubbles using US transducers at clinical frequencies is not feasible due to the comparatively long acoustic wavelengths employed. Here we present a technique which can be used to estimate nano- or microbubble size by virtue of the amount of motion detected in an M-Mode image acquired using an acoustic microscope equipped with a 200 MHz transducer. A sample of a bubble-containing solution is incorporated into a phantom composed of molten agarose. The solidified agarose gel contains pores with well-defined sizes dictated by the agarose concentration. Bubbles in the gel matrix that are smaller in diameter than the gel pore size are capable of undergoing stochastic motion which manifests as intensity fluctuations in M-Mode images. Conversely, bubbles which are larger than the agarose pores become trapped and produce static M-Mode intensity patterns. In this study, agarose gels with concentrations ranging from 0.25% to 1.25% (mean pore sizes ranging from 2.68 μm to 0.34 μm) were loaded with either nanobubbles (mean diameter 0.326 μm) or microbubbles (mean diameter 2.71 μm) and imaged at 200 MHz. In the nanobubble loaded gels, M-Mode fluctuations were clearly visible up to a gel concentration of 1% (pore size of 0.39 μm). In contrast, the microbubble loaded gels exhibited minimal M-Mode fluctuation even at agarose concentrations of 0.25% (2.68 μm pore size). Autocorrelation curves generated from the M-Mode data demonstrated a clear trend of curve flattening (loss of motion) when the pore size was comparable to mean bubble diameter, indicating that individual bubbles trapped in the agarose pores are the main source of acoustic backscatter. In the future, decay parameters extracted from the autocorrelation curves could potentially be used as indicators of mean bubble diameter for bubble populations of unknown size.

Nanoparticles have extensively been used for cancer therapy and imaging (i.e., theranostics) using various imaging modalities. Due to their physical and chemical properties (e.g., absorption, fluorescence, and magnetic properties) they have been used for image guided therapy for cancer treatment monitoring. There are various limitations that make many theranostic agents unable to be used for the extended periods of time required for enhancing theranostic capabilities. Some of these are due to inherent characteristics (e.g., change and/or breakdown of structure) present upon continuous irradiation and others are due to environmental (i.e., physiological) conditions that can lead to physical instability (i.e., in terms of size) affecting the amount of particles that can accumulate at the target site and the overall contrast that can be achieved. In this study, perfluorohexane (PFH) nanoemulsions (NEs) were synthesized with silica coated gold nanoparticles (PFH-NEs-scAuNPs) in order to give both stable and enhanced signals for cancer imaging by increasing vaporization of the emulsions into bubbles through the process of optical droplet vaporization (ODV). The resulting perfluorohexane bubbles could be imaged using nonlinear ultrasound (NL US) which significantly increases the signal to noise ratio due to the nonlinear scattering properties of oscillating bubbles. The NL US signals from PFH bubbles were found to be more stable compared to conventional bubbles used for contrast imaging. In addition, the vaporization of PFH NEs into bubbles was shown to cause significant cancer cell death reflecting the theranostic capabilities of the formed PFH bubbles. Since cell death is initiated with laser excitation of PFH-NEs-scAuNPs, these nanoparticles can specifically target cancer cells once they have accumulated at the tumor region. Due to the type of theranostic agent and imaging modality used, the PFH-NEs-scAuNPs can be used to provide higher specificity compared to other agents for locating the tumor region by minimizing tissue specific signals while at the same time being used to treat cancer.

The pressure threshold for 1/2 order subharmonic (SH) emissions and period doubling during the oscillations of ultrasonically excited bubbles is thought to be minimum when the bubble is sonicated with twice its resonance frequency (fr). This estimate is based on studies that simplified or neglected the effects of thermal damping. In this work, the nonlinear dynamics of ultrasonically excited bubbles is investigated accounting for the thermal dissipation. Results are visualized using bifurcation diagrams as a function of pressure. Here we show that, and depending on the gas, the pressure threshold for 1/2 order SHs can be minimum at a frequency between 0.5fr≤f≤0.6fr. In this frequency range, the generation of 1/2 order SHs are due to the occurrence of 5/2 order ultra-harmonic resonance. The stability of such oscillations is size dependent. For an air bubble immersed in water, only bubbles bigger than 1 μm in diameter are able to emit non-destructive SHs in these frequency ranges.

The pressure threshold for 1/2 order subharmonic (SH) emissions and period doubling during the oscillations of ultrasonically excited bubbles is thought to be minimum when the bubble is sonicated with twice its resonance frequency ( $f_{r}$ ). This estimate is based on studies that simplified or neglected the effects of thermal damping. In this work, the nonlinear dynamics of ultrasonically excited bubbles is investigated accounting for the thermal dissipation. Results are visualized using bifurcation diagrams as a function of excitation pressure. Here we show that contrary to the common belief, for an uncoated Air bubble, the pressure threshold for 1/2 order SHs is minimum at a frequency between .5 f_{r}\leq f\leq 0.6f_{r}$ . In this frequency range, the generation of 1/2 order SHs are due to the occurrence of 5/2 order ultra-harmonic resonance. The stability of such oscillations are size dependent. For an air bubble immersed in water, only bubbles bigger than $1\ \mu m$ in diameter are able to emit nondestructive SHs in these frequency ranges.

Purpose A real-time and non-invasive thermometry technique is essential in thermal therapies to monitor and control the treatment. Ultrasound is an attractive thermometry modality due to its relatively high sensitivity to change in temperature and fast data acquisition and processing capabilities. A temperature-sensitive acoustic parameter is required for ultrasound thermometry in order to track the changes in that parameter during the treatment. Currently, the main ultrasound thermometry methods are based on variation in the attenuation coefficient, the change in backscattered energy of the signal (CBE), the backscattered radio-frequency (RF) echo-shift due to change in the speed of sound and thermal expansion of the medium, and change in the amplitudes of the acoustic harmonics. In this work, an ultrasound thermometry method based on second harmonic CBE (CBEh2) and combined fundamental and second harmonic CBE (CBEcomb) is used to produce 2D temperature maps, detect localized heated region generated by low intensity focused ultrasound (LIFU), and control temperature in the heated region. Materials and methods Ex vivo pork muscle tissue samples were exposed to localized LIFU heating source and 2D temperature maps were produced from the RF data acquired by a 4.2 MHz linear array probe using a Verasonics Vantage™ ultrasound scanner (Verasonics Inc., Redmond, WA) after the exposure. Calibrated needle thermocouples were also placed in the ex vivo tissue sample close to the LIFU focal zone for temperature calibration purposes. The estimated temperature maps were the established echo-shift technique. A tissue motion compensation algorithm was also used to reduce the susceptibility to motion artifacts. Results 2D temperature maps were generated using CBE of acoustic harmonic and echo-shift techniques. The results show a direct correlation between the CBE of acoustic harmonics and focal tissue temperature for a range of temperatures from 37 °C (baseline) to 47 °C. Conclusions The findings of this study show that the CBE of acoustic harmonics technique can be used to noninvasively estimate temperature change in tissue in the hyperthermia temperature range.

The amount of gas in ultrasound contrast agents is related to their acoustic activity. Because of this relationship, gas volume has been used as a key variable in normalizing the in vitro and in vivo acoustic behavior of lipid shell-stabilized bubbles with different sizes and shell components. Despite its importance, bubble gas volume has typically only been theoretically calculated based on bubble size and concentration that is typically measured using the Coulter counter for microbubbles and nanoparticle tracking analysis (NTA) for nanoscale bubbles. However, while these methods have been validated for the analysis of liquid or solid particles, their application in bubble analysis has not been rigorously studied. We have previously shown that resonant mass measurement (RMM) may be a better-suited technique for sub-micron bubble analysis, as it can measure both buoyant and non-buoyant particle size and concentration. Here, we provide validation of RMM bubble analysis by using headspace gas chromatography/mass spectrometry (GC/MS) to experimentally measure the gas volume of the bubble samples. This measurement was then used as ground truth to test the accuracy of theoretical gas volume predictions based on RMM, NTA (for nanobubbles), and Coulter counter (for microbubbles) measurements. The results show that the headspace GC/MS gas volume measurements agreed well with the theoretical predictions for the RMM of nanobubbles but not NTA. For nanobubbles , the theoretical gas volume using RMM was 10% lower than the experimental GC/MS measurements, meanwhile, using NTA resulted in an 82% lower predicted gas volume. For microbubbles, the experimental gas volume from the GC/MS measurements was 27% lower compared to RMM and 72% less compared to the Coulter counter results. This study demonstrates that the gas volume of nanobubbles and microbubbles can be reliably measured using headspace GC/MS to validate bubble size measurement techniques. We also conclude that the accuracy of theoretical predictions is highly dependent on proper size and concentration measurements.

Different breakup regimes of bubbles and droplets caused by a sudden channel expansion in a microfluidic device are investigated. Without modifying the geometry and by only tuning several dimensionless parameters related to the fluid flow, a microchannel expansion region can produce mono-, bi-, or tri-disperse bubble or droplet populations.

Single sensor (pixel) signals require scanning of the sample in order to obtain spatial information. In this paper we show that using interference, optically induced signals can be reconstructed when recorded using interference pattern excitation, rather than a point illumination. This method reduces the need for dense scanning and requires a small number of scans, or can eliminate the need for scanning in some cases. It is shown that this method can be used in particular in 2D photo-acoustic imaging.

Phase change agents consisting of low boiling point perfluorocarbon (PFC) compounds have attracted increasing attention for ultrasound contrast-enhanced imaging and tumor therapy. However, the refraction, acoustic shadowing, reverberation, or limited penetration depth hamper their practical applications through previously reported acoustic droplet vaporization (ADV) or optical droplet vaporization (ODV) technique. Herein, we demonstrate a magnetic droplet vaporization (MDV) approach by loading perflurohexane (PFH) in magnetic mesoporous particles with a hollow space to carry out ultrasound imaging and tumor ablation. In vitro and in vivo magnetic thermal effects show that magnetic energy can be efficiently transformed into thermal energy by the PFH-encapsulated magnetic mesoporous particles, and then leading to vaporization of the loaded PFH. Owing to the generation of the PFH gas bubbles, the ultrasound signals are greatly improved in both harmonic mode and B mode. Simultaneously, anti-cancer experiments demonstrate that the tumor can be ablated after treating with the MDV method for 4 days, demonstrating highly efficient anti-cancer effects.

Microbubbles have applications in industry and life-sciences. In medicine, small encapsulated bubbles (< 10 μm) are desirable because of their utility in drug/oxygen delivery, sonoporation, and ultrasound diagnostics. While there are various techniques for generating microbubbles, microfluidic methods are distinguished due to their precise control and ease-offabrication. Nevertheless, sub-10 μm diameter bubble generation using microfluidics remains challenging, and typically requires expensive equipment and cumbersome setups. Recently, our group reported a microfluidic platform that shrinks microbubbles to sub-10 μm diameters. The microfluidic platform utilizes a simple microbubble-generating flow-focusing geometry, integrated with a vacuum shrinkage system, to achieve microbubble sizes that are desirable in medicine, and pave the way to eventual clinical uptake of microfluidically generated microbubbles. A theoretical framework is now needed to relate the size of the microbubbles produced and the system’s input parameters. In this manuscript, we characterize microbubbles made with various lipid concentrations flowing in solutions that have different interfacial tensions, and monitor the changes in bubble size along the microfluidic channel under various vacuum pressures. We use the physics governing the shrinkage mechanism to develop a mathematical model that predicts the resulting bubble sizes and elucidates the dominant parameters controlling bubble sizes. The model shows a good agreement with the experimental data, predicting the resulting microbubble sizes under different experimental input conditions. We anticipate that the model will find utility in enabling users of the microfluidic platform to engineer bubbles of specific sizes.

The study of blood coagulation can provide provide useful prognostic and diagnostic information for a range of diseases. Coagulation of whole mouse blood was monitored via a high-frequency ultrasound system with a central frequency of 80 MHz. Simultaneous acquisition of M-mode images and of reflected frequency spectra from signals propagated through the sample provided information on the motion of red blood cells and acoustic attenuation, respectively.This work aimed (i) to show the benefit of simultaneous acquisition of reflection spectra and M-mode data as blood coagulations, and (ii) to highlight the advantages of using high frequencies (> 60 MHz) in identifying features associated with blood coagulation which aren’t visible at lower frequencies.

We have developed a low-cost frequency domain differential photoacoustic microscope. The microscope uses a low-cost PA sensor made up of a kHz microphone, low power CW lasers and an optical chopper with 6/5 dual-slot disc. The system used dual-slot chopper disc to modulate two laser beams simultaneously and the corresponding PA signals were measured. This simultaneous modulation enabled a single scan instead of two scans required for two lasers in previous configurations, reducing the scanning time half. The configuration also enabled two PA interrogations at the same location of the sample whereas in the conventional sequential measuring system any backslash of the translation stage would alter the location of the interrogation area during the next measurement. The developed sensor is used to study the oxygen saturation of a single RBC. Two types of RBCs containing predominately oxy and methemoglobin were investigated using three different lasers of wavelengths 473 nm, 533 nm, and 633 nm. The single cell study showed that half of the oxy-RBC and meth-RBC exhibit oxygen saturation of about 90 % and 38.61 % respectively.

Optimization of the bubble performance requires size isolation and accurate shell characterization using models that are not limited by linear assumptions. MBs and NBs with 2 different shell compositions (crosslinked (C) and non-crosslinked (NC)) were made in-house. NC shell is made with 4 different lipids including DBPC, DPPA,DPPE and DSPE-PEG2000 . C shell bubbles have additional ingredients that produce a UV polymerized crosslinked shell. Using the method of multiple differential centrifugations, two distinct size populations were separated with mean diameters of 2.9 µm for NC-MB and 3.3 µm for C-MB. The attenuation and sound speed of the diluted solutions were measured through transmission and reception method using one pair of PVDF transducers with center frequencies of 10 MHz and 100% BW at acoustic pressures of approximately 5 to 40 kPa. Our nonlinear model accounting for large amplitude MB oscillations was used to fit the measured attenuation and sound speed data at each pressure. As the pressure increased from 5 kPa to ≈ 50kPa, resonance frequency (f r ) of the NC-MBs with a mean diameter (MD) of 2.9 µm decreased from 9.1 to 5.9 MHz and f r of the C-MBs with (MD) of 3.3 µm decreased from 8 to 4.8 MHz. NC-NB solutions did not display any attenuation peak in the frequency range of 2-10 MHz, additionally; the measured attenuation was 5-10 times smaller than the MBs with the same shell composition. Fitting of the shell parameters suggests that crosslinking the shell results in ≈ 37% increase in stiffness and 50 % decrease in shell viscosity. The lower attenuation of the NBs even at very high concentrations may explain the enhancement in NB contrast ultrasound.

Nanoemulsions have been used as theranostic agents for both imaging cancer, (using various imaging modalities) as well as for therapy through drug loading/release and vaporization. However, some of these imaging contrast agents are not stable enough to be used for long term monitoring of tumor growth/regression during therapy. When used in vivo, several nanoparticle agents rely on the use of optical absorbers which may have systemic toxicity. The current work uses perfluorohexane nanoemulsions (PFH-NEs) stabilized by a highly biocompatible optically absorbing fluorosurfactant shell that can be used to not only give stable PA signals but also enhance signals through the presence of long lasting perfluorohexane (PFH) bubbles formed after vaporization.

While the nucleus-to-cytoplasmic (N:C) ratio has traditionally been used for assessing cell malignancy, most N:C measurement techniques are time-consuming and performed on thin histological sections, which prohibit assessment of three-dimensional cell structure. A combined ultrahigh frequency ultrasound (US) and photoacoustic (PA) technique was used to assess the size and N:C ratio of cultured cancer cells in three dimensions (3D). The diameters of the cells and their stained nuclei were obtained by fitting the power spectrum of backscattered US pulses and emitted PA waves, respectively, to well-established theoretical models. For comparison, an imaging flow cytometer (IFC) was also used to determine the two-dimensional cell and nucleus sizes from large cell populations using brightfield and fluorescence images, respectively. An N:C ratio was calculated for each cell using the quotient of the measured nucleus diameter and the total cell diameter. The mean N:C ratios calculated using the sound-based approach were 0.68, 0.66, and 0.54 for MCF-7, PC-3, and MDA-MB-231 cells, respectively, and were in good agreement with the corresponding values of 0.68, 0.67, and 0.68 obtained using the IFC. The combined US and PA technique, which assesses cellular N:C ratio in 3D, has potential applications in the detection of circulating tumor cells in liquid biopsies.

Advancement of ultrasound molecular imaging applications requires not only a reduction in size of the ultrasound contrast agents (UCAs) but also a significant improvement in the in vivo stability of the shell-stabilized gas bubble. The transition from first generation to second generation UCAs was marked by an advancement in stability as air was replaced by a hydrophobic gas, such as perfluoropropane and sulfur hexafluoride. Further improvement can be realized by focusing on how well the UCAs shell can retain the encapsulated gas under extreme mechanical deformations. Here we report the next generation of UCAs for which we engineered the shell structure to impart much better stability under repeated prolonged oscillation due to ultrasound, and large changes in shear and turbulence as it circulates within the body. By adapting an architecture with two layers of contrasting elastic properties similar to bacterial cell envelopes, our ultrastable nanobubbles (NBs) withstand continuous in vitro exposure to ultrasound with minimal signal decay and have a significant delay on the onset of in vivo signal decay in kidney, liver, and tumor. Development of ultrastable NBs can potentially expand the role of ultrasound in molecular imaging, theranostics, and drug delivery.

Decellularization is a technique that permits the removal of cells from intact organs while preserving the extracellular matrix (ECM). It has many applications in various fields such as regenerative medicine and tissue engineering. This study aims to differentiate between fresh and decellularized kidneys using quantitative ultrasound (QUS) parameters. Spectral parameters were extracted from the linear fit of the power spectrum of raw radio frequency data and parametric maps were generated corresponding to the regions of interest, from which four textural parameters were estimated. The results of this study indicated that decellularization affects both spectral and textural parameters. The Mid Band Fit mean and contrast were found to be the best spectral and textural predictors of kidney decellularization, respectively.

Opto-acoustic imaging involves using light to produce sound waves for visualizing blood in biological tissue. By using multiple optical wavelengths, diagnostic images of blood oxygen saturation and total hemoglobin are generated using endogenous optical contrast, without injection of any external contrast agent and without using any ionizing radiation. The technology has been used in recent clinical studies for diagnosis of breast cancer to help distinguish benign from malignant lesions, potentially reducing the need for biopsy through improved diagnostic imaging accuracy. To enable this application, techniques for mapping oxygen saturation differences within tissue are necessary. Using biologically relevant opto-acoustic phantoms, we analyze the ability of an opto-acoustic imaging system to display colorized parametric maps that are generated using a statistical mapping approach. To mimic breast tissue, a material with closely matching properties for optical absorption, optical scattering, acoustic attenuation, and speed of sound is used. The phantoms include two vessels filled with whole blood at oxygen saturation levels determined using a sensor-based approach. A flow system with gas-mixer and membrane oxygenator adjusts the oxygen saturation of each vessel independently. Datasets are collected with an investigational Imagio® breast imaging system. We examine the ability to distinguish vessels as the oxygen saturation level and imaging depth are varied. At depth of 15 mm and hematocrit of 42%, a sufficient level of contrast to distinguish between two 1.6-mm diameter vessels was measured for an oxygen saturation difference of ∼4.6%. In addition, an oxygenated vessel was visible at a depth of 48 mm using an optical wavelength of 1064 nm, and a deoxygenated vessel was visible to a depth of 42 mm with 757 nm. The results provide insight toward using color mapped opto-acoustic images for diagnosing breast cancer.

Advancement of ultrasound molecular imaging applications requires not only a reduction in size of the ultrasound contrast agents (UCAs) but also a significant improvement in the in vivo stability of the shell-stabilized gas bubble. The transition from first generation to second generation UCAs was marked by an advancement in stability as air was replaced by a hydrophobic gas, such as perfluoropropane and sulfur hexafluoride. Further improvement can be realized by focusing on how well the UCAs shell can retain the encapsulated gas under extreme mechanical deformations. Here we report the next generation of UCAs for which we engineered the shell structure to impart much better stability under repeated prolonged oscillation due to ultrasound, and large changes in shear and turbulence as it circulates within the body. By adapting an architecture with two layers of contrasting elastic properties similar to bacterial cell envelopes, our ultrastable nanobubbles (NBs) withstand continuous in vitro exposure to ultrasound with minimal signal decay and have a significant delay on the onset of in vivo signal decay in kidney, liver, and tumor. Development of ultrastable NBs can potentially expand the role of ultrasound in molecular imaging, theranostics, and drug delivery.

In photoacoustic (PA) imaging, time domain reconstruction techniques are the current gold standard for image formation. While these techniques provide high-resolution spatial maps of optical absorption, they neglect the structural information encoded in the frequency domain of the broadband PA signals. In this work, we introduce a frequency domain technique for PA image formation, termed F-Mode. By leveraging information contained in the frequency content of PA signals, F-Mode can be used to generate images with scale-specific contrast. To demonstrate the robustness of our technique, we apply F-Mode to datasets acquired using both PA tomography and PA microscopy systems, utilizing linear array and single-element transducers with central frequencies ranging from 40–400 MHz. Here we show that the technique can be used to: differentiate between vessels and microspheres of different size in phantoms, enhance visualization of organelles in cultured cells, and selectively display single blood vessels in vivo in zebrafish larvae. Standard time domain photoacoustic imaging techniques neglect the abundant information encoded in the frequency domain of photoacoustic signals. The authors present an imaging technique that utilizes features in photoacoustic signal power spectra to visualize structures of different scale in image datasets acquired using classical methods.

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.

In this work, speckle in acoustic-resolution photoacoustic (PA) imaging systems is discussed. Simulations and experiments were used to demonstrate that PA speckle carries structural information related to sub-resolution absorbers. Numerical simulations of phantoms containing spherical absorbers were performed using Green’s function solutions to the PA wave equation. A 21 MHz linear array was simulated (256 elements, 75×165 µm resolution, bandwidth 9-33 MHz) and used to record, bandlimit and beamform the generated PA signals. The effects of absorber size (10-270 µm) and concentration (10-1000/mm3) on PA speckle were examined using envelope statistics and radiofrequency spectroscopy techniques. To examine PA speckle experimentally, a VevoLAZR system was used to image gelatin phantoms containing 3 and 15µm polystyrene beads, a tissue mimicking radial artery phantom, and murine tumour vasculature in vivo. Fully developed speckle, as assessed by Rayleigh distribution fits to PA signal envelopes, was present in all images (simulated and experimental) containing at least 10 absorbers per resolution volume, irrespective of absorber size. Changes in absorber size could be detected using the spectral slope of the normalized power spectrum (4.5x decrease for an 80 µm increase in size). PA images of flowing blood in the radial artery phantom also revealed the presence of speckle with intensity that fluctuated periodically with beat rate (4 dB per cycle). Speckle was ubiquitous to all murine tumor vasculature images. During treatment-induced vascular hemorrhaging, the spectral slope decreases by 80% compared to untreated mice. These results demonstrate that photoacoustic speckle encodes information about the underlying absorber distribution.

Opto-acoustic imaging involves using light to produce sound waves for visualizing blood in biological tissue. By using two different optical wavelengths, diagnostic images of blood oxygen saturation can be generated using endogenous optical contrast, without injection of any external contrast agent and without using any ionizing radiation. The technology has been used in recent clinical studies for diagnosis of breast cancer to help distinguish benign from malignant lesions, potentially reducing the need for biopsy through improved diagnostic imaging accuracy. To enable this application, techniques that can accurately map and effectively display small differences in oxygen saturation are necessary. We analyze the ability of an opto-acoustic imaging system to display a colorized parametric map for oxygen saturation of blood using biologically-relevant opto-acoustic phantoms. The relationship between colorized image output and known oxygen saturation values is examined. To mimic breast tissue, a material with closely matching properties for optical absorption, optical scattering, acoustic attenuation and speed of sound is used. The phantoms include two vessels filled with whole blood at oxygen saturation levels determined using a gold-standard sensor-based approach. A flow system with gas-mixer and membrane oxygenator adjusts the oxygen saturation of each vessel independently. Data is collected with an investigational Imagio breast imaging system. An opto- acoustic relative color map is generated using a novel statistical mapping approach. In addition, we propose a technique to characterize the ability to distinguish small differences in oxygen saturation as the oxygenation level is varied. When applied to the phantom, with reference vessel at 99% saturation, hematocrit of 42% and depth of 1.5cm, a contrast distinction threshold was reached when an adjusted vessel achieved a difference of approximately 4.6% saturation compared to the reference.

A photoacoustic sensor for studying the thermal properties of a single biological cell was developed. The sensor used an aluminium foil for heating the sample which enables the study of unstained samples. The PA sensor was developed to work in the heat transmission mode configuration by positioning the detector at the opposite side with respect to heated side of the sample. Rosencwaig’s theoretical model for an open PA cell configuration functional in a heat transmission mode was applied to study the thermal diffusivity of biological cells. MCF7 cells were used in this studied. We measured the thermal diffusivity of 3 cells using the sensor. The average measured thermal diffusivity of MCF7 cells was 0.05 mm 2 /s. The PA sensor allows the spatial mapping of the cell thermal diffusivity and can be used to measure thermal properties of cells in various phases of the cell cycle. It can also be used to measure the effective properties of cells when they have internalized various sensors (e.g. plasmonic nanoparticles).

A cell’s nucleus-to-cytoplasm (N:C) ratio is a histological metric used to stage malignant disease. Current N:C assessment methods, such as optical microscopy, are time-consuming, subjective, and low-throughput. Here, we compare the N:C ratios of prostate cancer (PC-3) cells measured by a novel microfluidic PhotoAcoustic Flow Cytometer (PAFC) to those obtained using an Imaging Flow Cytometer (IFC). PC-3 cells were stained with DRAQ-5 nuclear dye and divided into populations measured using the PAFC and IFC. The PAFC consisted of a microfluidic device integrated with a singleelement ultrasound transducer (375 MHz central frequency) and a sub-nanosecond pulsed laser (532 nm). Individual cells were 3D flow-focused through the overlapping focal region of the ultrasound and laser pulses. PAFC estimation of the cell and nucleus diameters were determined through power spectra fitting of backscattered US waves and emitted PA waves to established theoretical models. An ImageStreamX® IFC was used to acquire brightfield and fluorescent images of individual cells, which were masked, gated, and used to assess the cell (brightfield) and nucleus (fluorescence) diameter to validate the PAFC measurements. The average cell and nucleus diameters determined using the PAFC (n = 388) were 18.8 ± 3.3 μm and 14.3 ± 2.9 μm, respectively. The corresponding values from the IFC (n = 4651) were 18.3 ± 2.2 μm and 12.2 ± 1.9 μm. The N:C ratio (calculated as the ratio of the nucleus diameter to cell diameter) was 0.77 ± 0.10 using the PAFC and 0.67 ± 0.07 using the IFC. Our novel PAFC device has the potential to be used for circulation tumor cell detection using the N:C ratios of cells.

We use a novel acoustic-based flow cytometer to detect individual nanobubbles flowing in a microfluidic channel using high-frequency ultrasound and photoacoustic waves. Each individual nanobubble (or cluster of nanobubbles) flowing through the foci of high-frequency ultrasound (center frequency 375 MHz) and nanosecond laser (532 nm) pulses interacts with both pulses to generate ultrasound backscatter and photoacoustic waves. We use in-house generated nanobubbles, made of lipid shells and octafluoropropane gas core, to detect ultrasound backscatter signals using an acoustic flow cytometer. Nanobubble solutions sorted in size through differential centrifugation are diluted to 1:10,000 v/v in phosphate buffered saline solution to maximize the probability that the detected signals are from individual nanobubbles. Nanobubble populations were sized using resonant mass measurement. Results show that the amplitude of the detected ultrasound backscatter signal is dependent on the nanobubble size. The average amplitude of the ultrasound backscatter signals from at least 950 nanobubbles with an average diameter of 150 nm, 225 nm, and 350 nm was 5.1±2.5 mV, 5.3±2.3 mV, and 6.4±1.8 mV, respectively. Similarly, we detected interleaved ultrasound backscatter and photoacoustic signals from nanobubbles tagged with Sudan Black B dye. The average amplitude of the ultrasound backscatter and photoacoustic signals from these black nanobubbles with an average diameter of 238 nm is 10±11 mV and 54±75 mV, respectively. The presence of the dye on the shell suppressed unique features seen in the ultrasound backscatter from the nanobubbles without dye. At present, there is no robust commercial technique able to analyze the ultrasonic response of individual nanobubbles. The acoustic flow cytometer can potentially be used to analyze physical parameters, such as size and ultrasonic response, of individual nanobubbles.

A cell’s nucleus-to-cytoplasm ratio (N:C) can be used as a metric in histology for staging malignancy. Current techniques used for N:C assessment are time-consuming, low-throughput, and lack 3-D structure assessment. In this study, we assess the N:C ratio of MCF-7 cells using an ultra-high frequency acoustic/photoacoustic (PA) microscope and compare measurements to an imaging flow cytometer (IFC). MCF-7 cells were stained with the DRAQ-5 nuclear dye to facilitate production of fluorescence and PA signals. A PA microscope (PAM) consisting of a 375 MHz transducer and a pulsed 532 nm laser focused through a 10X optical objective was used for cell measurements. Cell and nucleus diameters of 37 stationary MCF-7 cells were obtained by fitting the power spectrum of backscattered ultrasound pulses and emitted PA waves, respectively, to well-established theoretical models. An Amnis ImageStreamX® IFC acquired brightfield/fluorescence images of cells and their nuclei, respectively. Masking and gating techniques were used to obtain the cell and nucleus diameters for 2004 MCF-7 cells from the IFC. For both systems, the N:C ratio was calculated as the ratio ofthe nucleus diameter to total cell diameter. The average cell and nucleus diameters from PAM were 15.2 ± 3.5 μm and 10.2 ± 3.5 μm, respectively, and were in good agreement with the average cell and nucleus diameters of 18.7 ± 2.6 μm and 12.6 ± 1.9 μm measured by the IFC. The N:C ratios were in excellent agreement, calculated to be 0.68 ± 0.19 and 0.68 ± 0.09 for PAM and IFC, respectively. This study demonstrates the ability of PAM to measure the N:C ratio of cells which in excellent agreement with IFC assessments.

Highlights • Bifurcation structure of the lipid coated microbubbles (MBs) is studied • Effect of the coating is analyzed by comparing the dynamics of the lipid coated MBs to uncoated ones. • Buckling and rupture of the lipid coating enhances the nonlinear oscillations. • Period 2, Period 3 or chaos can be generated at pressures as low as 1 kPa. • In addition to subharmonic emissions, superharmonic emissions may also be enhanced. • With increasing pressure, period 2 is generated, then disappears and is regenerated again., In many applications, microbubbles (MBs) are encapsulated by a lipid coating to increase their stability. However, the complex behavior of the lipid coating including buckling and rupture sophisticates the dynamics of the MBs and as a result the dynamics of the lipid coated MBs (LCMBs) are not well understood. Here, we investigate the nonlinear behavior of the LCMBs by analyzing their bifurcation structure as a function of acoustic pressure. We show that, the LC can enhance the generation of period 2 (P2), P3, higher order subharmonics (SH), superharmonics and chaos at very low excitation pressures (e.g. 1 kPa). For LCMBs sonicated by their SH resonance frequency and in line with experimental observations with increasing pressure, P2 oscillations exhibit three stages: generation at low acoustic pressures, disappearance and re-generation. Within non-destructive oscillation regimes and by pressure amplitude increase, LCMBs can also exhibit two saddle node (SN) bifurcations resulting in possible abrupt enhancement of the scattered pressure. The first SN resembles the pressure dependent resonance phenomenon in uncoated MBs and the second SN resembles the pressure dependent SH resonance. Depending on the initial surface tension of the LCMBs, the nonlinear behavior may also be suppressed for a wide range of excitation pressures.