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Aluminium sulphonated phthalocyanine (AlPs) photosensitiser mixed with Hemoglobin (Hb) was used to create patterned oxygenation states of Hb in tissue mimicking gelatin phantoms. Temporal study of Hb conversion and an oxygenation patterned phantom is presented.

Synthetic transmit aperture (STA) imaging systems usually have a lower SNR compared with conventional Bmode ultrasound systems because only one or a small number of elements are selected for each transmission in STA. Here we propose delay-encoded synthetic transmit aperture (DE-STA) imaging to encode all the transmission elements to increase the SNR of the pre-beamformed RF signals. The encoding scheme is similar to the Hadamard encoding. However, in each transmission of DE-STA imaging, selected transmitting elements are delayed by a half period of the ultrasound wave relative to the rest transmitting elements, rather than using a pulse inversion as in the Hadamard encoding sequence. After all the transmission events, a decoding process in the temporal frequency domain is applied to the acquired RF signals to recover the equivalent traditional STA signals with a better SNR. The proposed protocol is tested with simulated data (using Field II) and experimental data acquired with a commercial linear array imaging system (Ultrasonix RP). The results from both the simulations and the experiments demonstrate increased SNR of pre-beamformed RF signals and improved image quality in terms of peak signal-to-noise ratio (PSNR), resolution and contrast-to-noise ratio compared with traditional STA. The lateral resolution (as assessed by a wire target) of DESTA imaging is improved by 28% and the PSNR of the wire is increased by 7 dB, respectively, compared with traditional STA imaging. The proposed image reconstruction framework can also be extended to other transmission protocols.

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

Hyperthermia is a cancer treatment technique that could be delivered as a stand-alone modality or in conjunction with chemotherapy or radiation therapy. Noninvasive and real-time temperature monitoring of the heated tissue improves the efficacy and safety of the treatment. A temperature-sensitive acoustic parameter is required for ultrasound-based thermometry. In this paper the amplitude and the energy of the acoustic harmonics of the ultrasound backscattered signal are proposed as suitable parameters for noninvasive ultrasound thermometry.A commercial high frequency ultrasound imaging system was used to generate and detect acoustic harmonics in tissue-mimicking gel phantoms and ex vivo bovine muscle tissues. The pressure amplitude and the energy content of the backscattered fundamental frequency (p1 and E1), the second (p2 and E2) and the third (p3 and E3) harmonics were detected in pulse-echo mode. Temperature was increased from 26° to 46 °C uniformly through both samples. The amplitude and the energy content of the harmonics and their ratio were measured and analysed as a function of temperature.The average p1, p2 and p3 increased by 69%, 100% and 283%, respectively as the temperature was elevated from 26° to 46 °C in tissue samples. In the same experiment the average E1, E2 and E3 increased by 163%, 281% and 2257%, respectively. A similar trend was observed in tissue-mimicking gel phantoms.The findings suggest that the harmonics generated due to nonlinear ultrasound beam propagation are highly sensitive to temperature and could potentially be used for noninvasive ultrasound tissue thermometry.

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.

Through Silicon Via (TSV) is the most promising technology for vertical interconnection in novel three-dimensional chip architectures. Reliability and quality assessment necessary for process development and manufacturing require appropriate non-destructive testing techniques to detect cracks and delamination defects with sufficient penetration and imaging capabilities. The current paper presents the application of two acoustically based methods operating in the GHz-frequency band for the assessment of the integrity of TSV structures.

We investigate the optical wavelength dependence in quantitative photoacoustic (QPA) assessment of red blood cell (RBC) aggregation and oxygen saturation (sO2 ) during pulsatile blood flow. Experimentally, the pulsatile flow was imaged with a 700 to 900 nm laser using the VevoLAZR. Theoretically, the photoacoustic (PA) signals were computed based on a Green's function integrated with a Monte Carlo simulation of radiant fluence. The pulsatile flow created periodic conditions of RBC aggregation/nonaggregation, altering the aggregate size, and, in turn, the sO2 . The dynamic range, DR (a metric of change in PA power) from 700 to 900 nm for nonaggregated RBCs, was 5 dB for both experiment and theory. A significant difference in the DR for aggregated RBCs was 1.5 dB between experiment and theory. Comparing the DR at different wavelengths, the DR from nonaggregated to aggregated RBCs at 700 nm was significantly smaller than that at 900 nm for both experiment (4.0 dB < 7.1 dB) and theory (5.3 dB < 9.0 dB). These results demonstrate that RBC aggregation simultaneously affects the absorber size and the absorption coefficient in photoacoustic imaging (PAI) of pulsatile blood flow. This investigation elucidates how QPA spectroscopy can be used for probing hemodynamics and oxygen transport by PAI of blood flow.

Ultrasonic backscatteredsignals contain information regarding the scatterer structures of the imaged biological tissues; a uniform scatterer distribution could be represented by periodicities in the backscattered signals. This work aims to characterize these scatterer periodicities using wavelet improved cepstral analysis. This technique was tested on simulated ultrasound signals, where the periodicity was clearly visible. Simulation results indicate that this technique can effectively determine the value of the scatterer spacing. The technique was then tested on a HT-29 cell pellet, where the estimated scatterer spacing was found to be 17.67 ± 3.85 μm. Future work includes improving the technique with the aim of accurately estimating the mean scatterer spacing in tissues.

Systemic chemotherapy has been associated with poor tumor penetration and adverse cytotoxic effects. To combat this, cancer therapy with in-situ forming implants (ISFIs) can provide continuous, high dose release of chemotherapeutic drug directly at the tumor site. However, poor drug distribution through the tumor volume has limited the effectiveness and subsequent translation of ISFIs into clinical practice. Our lab has previously demonstrated an increase in drug release and penetration from injectable, phase inverting PLGA implants in an in-vitro phantom model using low frequency ultrasound. To build on this work, in-vivo fluorescein intensity and distribution was evaluated in subcutaneous ISFIs with and without therapeutic ultrasound (TUS) exposure.

We present a new technique for photoacoustic (PA) image formation, termed ‘F-Mode’, which capitalizes on variations in the power spectrum of PA signals to produce images with object specific contrast. The technique is applied to a PA dataset by calculating the signal power spectrum at each scan location, segmenting it into discrete frequency bands, and then forming an image representing the spatial power distribution for each band. The appearance of differently sized objects in the resultant F-Mode images is dynamic, and is dictated by the presence of structure specific features in the power spectra. To demonstrate the technique, black polystyrene microspheres with diameters of 6 and 10 μm were scanned using a PA microscope equipped with a 400 MHz transducer and 532 nm laser. The images demonstrated that with appropriate selection of frequency band, visualization of either population of spheres could be selectively enhanced; the 6 μm spheres being more prominent at 249 MHz, while the 10 μm spheres dominated in the 425 MHz F-Mode image. Further, unique frequency dependent patterning in images of individual spheres pointed towards sub-micron diameter fluctuations in spheres from the same population. This proof-of-concept work paves the way for future in vivo applications, such as selectively analyzing blood vessels of different diameters.

Nanoscale ultrasound agents have recently gained interest in cancer imaging and therapy for their ability to exploit the enhanced permeability and retention (EPR) effect in tumors. There have been numerous reports of echogenic nanoscale bubbles, but it remains unclear whether these sub-micron bubbles are responsible for producing the observed acoustic backscatter at clinically relevant frequencies. Because nanobubble polydispersity is high, larger microbubbles are thought to be the significant contributors to the observed signal. Accordingly, this study examined echogenicity of lipid- and surfactant-stabilized perfluoropropane nanobubbles following extra steps to ensure larger bubbles were removed from solution. Microbubbles were separated from nanobubbles based on their buoyancy by centrifugation at 50g for 5 min. Isolated nanobubbles were also filtered using track-etched polycarbonate (PCTE) filters with a pore size of 1.0 micron, 0.6 micron, and 0.4 micron using a syringe pump at 100 microliters/min and were imaged in a custom agarose mold in PBS using contrast harmonic imaging (Toshiba, 12 MHz, MI 0.1). Nanobubbles isolated via centrifugation were found to have considerable echogenicity at 12 MHz. Resonant mass measurement results indicate that these bubbles were below 1 micron in diameter and typical size range was between 100 to 600 nm (mean diameter 233 ± 5 nm). A reduction in signal after filtration was noted, but NB activity was observed under all conditions. This study demonstrates that sub-micron bubbles are capable of producing strong echogenic signals at clinically used ultrasound frequencies.

In situ forming implants (ISFIs) can provide continuous, high dose release of chemotherapeutic drug directly at the tumor site. However, poor drug distribution through the tumor volume has limited the effectiveness and subsequent translation of ISFIs into clinical practice. In this study, mice were subcutaneously injected with phase-inverting ISFIs loaded with fluorescein and exposed to therapeutic ultrasound (TUS) at varying parameters to evaluate fluorescein distribution and release. An increased spatial penetration of fluorescein away from the ISFIs in vivo was linked to increased temperature from the TUS application. Without the increased temperature, ISFIs exposed to a similar amount of total TUS energy saw no significant increase in fluorescein distribution compared to untreated ISFIs. Surprisingly, the total amount of fluorescein released from the ISFIs was consistent among all groups. The observed increase in fluorescein distribution may thus be due to a hyperthermia-induced increase in cell membrane permeability that allows nearby cells to more readily uptake fluorescein. The US-enhanced distribution could improve the clinical efficacy of intratumoral chemotherapy by increasing drug distribution through the tumor volume without increasing drug release from the implant.

In this study, a phase-change contrast agent made of biodegradable polymer PLGA and loaded with gold nanorods (GNRs) and perfluorohexane were conjugated to an anti-HER2 antibody (Herceptin) for specific binding to breast cancer cells that overexpress HER2 receptors. The mean size of the nanoparticles (NPs) was about 500 nm. The targeting specificity of PLGA-GNR-Herceptin NPs was examined using fluorescent imaging, flow cytometry, and photoacoustic (PA) imaging. Flow cytometry results demonstrated that 53% DiD-positive staining was found in HER2-positive BT474 cells after incubation with nanoparticle/antibody conjugates for one hour, while only 3.8% DiD-positive staining was found in HER2-negative MDA-MB-231 cells, indicating receptor-specific binding of the conjugated PLGA NPs. This binding was confirmed by fluorescent imaging. The PA image enhancement with a 19-fold increase in the signal-to-noise ratio was also demonstrated in vitro using targeted NPs. This particle can be loaded with chemo drugs and may be a potential cancer biomarker and therapeutic agent using PA imaging guidance.

One of the main advantages of photoacoustic (PA) microscopy is its ability to generate high-resolution maps of vasculature due to strong endogenous absorption. In contrast the optical absorption of the gross anatomy surrounding the vasculature is negligible, and it is thus not readily imaged using PA. We have previously reported on a new imaging technique, termed Photoacoustic Radiometry (PAR), which derives contrast from the sample's optical attenuation properties (doi:10.1117/12.2212961). In this work, we use a dual PA and PAR technique to simultaneously acquire co-registered images of the vasculature and gross anatomy of the trunk of a zebrafish embryo from a single PA scan.

Imaging speckle arises from the interference of waves from randomly distributed sources. Here, we provide a theoretical basis and experimental evidence for the presence of speckle in photoacoustic (PA) imaging across multiple ultrasonic (US) detection frequencies.

Our group has previously demonstrated that lower shear rate in the pulsatile blood flow yields greater red blood cell (RBC) aggregation which in turn results in a higher oxygen saturation (sO 2 ) level and a higher photoacoustic (PA) signal. Higher shear rates led to disaggregation thereby decreasing the PA signal amplitude and the sO 2 . These results suggest that the interrelationship between the sO 2 and RBC aggregation may provide a new biomarker in the diagnosis of diseases that alter blood rheological properties. In this paper, we present a pilot study where high-frequency photoacoustic imaging (PAI) is used for the simultaneous assessment of RBC aggregation and sO 2 in vivo in the human radial artery (RA). The ultrasound (US) and PA images in the RA were acquired using a US/PA imaging system equipped with a 21 MHz linear-array probe (Vevo LAZR; LZ250, FUJIFILM VisualSonics, Canada), varying the wavelength of optical illumination (700, 750, 800, 850 and 900 nm). The blood flow velocity at the RA was assessed by pulsed wave Doppler in the same device. The PA signals inside the RA were observed at all wavelengths. The PA power increased with the illumination wavelength. At each wavelength, the PA power varied as function of time. The phase of variation in PA signals was inversely proportional to that in systolic blood flow velocity. The sO 2 is proportional to the cell surface area exposed to the surrounding media, and RBC aggregation decreases the exposed area of the aggregate. As such, the sO 2 was higher for aggregated cells (diastolic state) than single cells (systolic state). This PAI study of RA RBC aggregation and sO 2 in vivo is the first attempt to study the hemodynamics and physiological function of RBC, which can be used as a potential tool for the diagnosis of blood flow conditions in the RA.

Medical imaging through a targeting agent promises to improve the specificity and sensitivity of cancer detection. Nanoparticles (NPs) may provide advantages for targeted photoacoustic (PA) imaging due to their unique physical and optical properties. In our previous study, we developed a phase-change contrast agent made from a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA) and loaded with perfluorohexane (PFH) liquid, gold NPs and fluorescent dyes for PA imaging and cancer therapy [1]. In this study, The PLGA NPs were conjugated to an anti-HER2 antibody (Herceptin) for specific binding to breast cancer cells that overexpress HER2 receptors. The targeting specificity of PLGA-GNR-Herceptin NPs and the resultant PA enhancement in cells were examined using fluorescent imaging, flow cytometry, and PA imaging.

The usage of microbubbles (MBs) is limited to the blood pool due to their large size yet the detection of biomarkers on tumor cells and effective drug delivery, require MBs to reach the tumor tissue outside of the vasculature. To tackle these problems, nanobubbles (NBs) are proposed as a potential alternative. NBs can pass through submicron blood vessels and extravasate to tissue. Due to their higher number density; higher doses of NBs can be delivered to the target. However, despite their potential, the use of NBs has been limited because of the limited information of their complex dynamics. In this work, we manufactured lipid and surfactant-stabilized C3F8 NBs (mean diameter ∼200 nm). NB scattering response was investigated by single bubble scattering experiments with narrowband pulses with 16–55 MHz and acoustic pressure of 0.250–1.5 MPa (Vevo-770 Machine, Fujifilm visualsonics), and in-vivo imaging at 18 MHz and 4% power (Vevo 3100, Fujifilmvisualsonics). The nonlinear response of the NBs was numerically studied by solving the Marmottant model for the US pulses used in the experiments. The results were visualized using the resonance curves and bifurcation diagrams of the oscillations of the NBs versus frequency and pressure. Experimental results demonstrate strong echogenicity of NBs at a frequency range of 10–25 MHz. Single NB experiments suggest that NBs generate strong subharmonic and super harmonic responses even at lower acoustic pressures ∼250 kPa. This contradicts the linear theoretical predictions, as the resonance frequency (fr) of the NBs is calculated to be ∼130 MHz. Results of numerical simulations show that when the initial surface tension of the NBs is ∼

Understanding of the propagation of ultrasound through a bubbly medium is a challenging task because of the nonlinear dynamics of the bubbles and their effect on the attenuation and sound speed of the medium. The majority of the studies on this subject apply linear models, which will generate inaccurate results, especially at higher-pressure excitations. These studies have also ignored the effect of bubble-bubble interaction and nonlinear multiple scattering. In this work, we have numerically simulated the attenuation and sound speed of a bubbly medium by solving our recently developed nonlinear model. An efficient method to investigate the nonlinear bubble-bubble interaction and multiple scattering is developed, and this phenomenon is included the numerical investigations through considering a cluster of 130 randomly distributed interacting bubbles with sizes derived from experimental measurements. Broadband experimental attenuation measurements of monodisperse lipid-coated microbubble solutions were performed with peak acoustic pressures ranging within 10–100kPa. The bubble solutions had mean diameters of 4–6 micron and peak concentrations of 1000 to 15000 bubbles/ml. At lower concentrations (with minimal bubble-bubble interactions), predictions of the model (attenuation and sound speed vs frequency) in the absence of interaction are in good agreement with experimental measurements. At higher concentrations, secondary peaks in the attenuation and sound speed diagrams as a function of frequency appear. Through considering the bubble-bubble interactions, the numerical results can predict the quantitative and qualitative changes in the attenuation and frequency as well as the generation of secondary peaks.

Photoacoustic (PA) imaging has been proposed for cancer treatment monitoring. Tumor oxygen saturation (sO 2 ) should in principle be related to vascular parameters such as blood flow. In this work, in-vivo PA estimates of sO 2 were compared to power Doppler (pD) measures of vascularity hours after the administration of microbubbles (MB), radiation therapy (XRT), individually or combined (MB-XRT).

Most chemotherapeutics (CTs) are delivered systemically, causing nausea, hair loss, fatigue and a compromised immune system. Biocompatible in situ forming implants (ISFIs) are drug delivery vehicles which are injected as a liquid before solidifying in tissues and ultimately breaking down. By dissolving CTs in an ISFI solution, they can be injected directly to the tumour site and released in a controllable manner. ISFIs can provide localized, continuous release of CT, reducing side effects. The complex phase change of ISFIs causes a variable release rate of CT. In this work, photoacoustic (PA) imaging was used for the first time to monitor a dye (mimicking CT) diffusing into a tissue mimicking phantom while quantitative ultrasound (QUS) was used to monitor the changes in the ISFI structure. ISFIs made of poly(lactic-co-glycolic acid) and Janus Green B dye dissolved in N-methyl-2-pyrrolidone in a 39:1:60 ratio were injected in tissue mimicking polyacrylamide phantoms containing titanium oxide. ISFI structure and drug release was monitored over 72 hours. At each time point, 47 planes of the phantoms were imaged using both PA (700nm) and US using the VevoLAZR system. Regions of interest within and proximal to the ISFI were selected, and average PA and QUS parameters were determined for each plane as a function of time postimplantation. This work shows the potential of PA and QUS for monitoring kinetic drug release.

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.

In this chapter, we describe two new methodologies: (1) application of high-frequency ultrasound spectroscopy for in vivo detection of cancer cell death in small animal models, and (2) extension of ultrasound spectroscopy to the lower frequency range (i.e., 1-10 MHz range) for the detection of cell death in vivo in preclinical and clinical settings. Experiments using tumor xenografts in mice and cancer treatments based on chemotherapy are described. Finally, we describe how one can detect cancer response to treatment in patients noninvasively early (within 1 week of treatment initiation) using low-frequency ultrasound spectroscopic imaging and advanced machine learning techniques. Color-coded images of ultrasound spectroscopic parameters, or parametric images, permit the delineation of areas of dead cells versus viable cells using high ultrasound frequencies, and the delineation of areas of therapy response in patient tumors using clinically relevant ultrasound frequencies. Depending on the desired resolution, parametric ultrasound images can be computed and displayed within minutes to hours after ultrasound examination for cell death. A noninvasive and express method of cancer response detection using ultrasound spectroscopy provides a framework for personalized medicine with regards to the treatment planning of refractory patients resulting in substantial improvements in patient survival.

High-frequency ultrasound (20 MHz) spectroscopy can be used to detect noninvasively DNA damage in cell samples in vitro, and in live tissue both ex vivo and in vivo. This chapter focuses on the former two aspects. Experimental evidence suggests that morphological changes that occur in cells undergoing apoptosis result in changes in frequency-dependent ultrasound backscatter. With advances in research, ultrasound spectroscopy is advancing the boundaries of fast, label-free, noninvasive DNA damage detection technology with potential use in personalized medicine and early therapy response monitoring. Depending on the desired resolution, parametric ultrasound images can be computed and displayed within minutes to hours after ultrasound examination for cell death.

Photoacoustic or PA waves, generated from blood cells, create distinct spectral features in the Fourier domain, for example, maxima and minima. In this way, high-frequency PA signals can be used to identify and distinguish blood cells. However, due to finite bandwidth of physical systems, many interesting Fourier features are invisible within the observed bandwidth. To overcome this challenge, we reformulate the PA imaging problem as a time-of-flight super-resolution problem. Based on the PA wave equations, we show that the problem reduces to estimation of sparse cellular features from a set of finite trigonometric moments. For this purpose, we develop a super-resolution algorithm which achieves near exact performance (in context of maximum likelihood estimation) when working with experimental data. Hence, our work alleviates an important bottleneck in PA imaging linked with classification of cellular features.

Photoacoustic (PA) imaging of tumor oxygenation can be used to monitor vascular-targeted novel therapies. This study examines how a combination treatment, ultrasound-microbubbles (USMB)/radiation-therapy (XRT) alters oxygen saturation (sO2) estimates, which are then compared to power Doppler (PD) assessments of tumor vascularity. SCID mice were inoculated with subcutaneous, hind-leg PC3 tumors. The treatment consisted of XRT/MB (XRT: 8Gy/single-fraction; USMB: 3%/500 kHz/570kPa; n=3), USMB (n=3) and XRT (n=5) alone and untreated control (n=5). PA/PD imaging was acquired pre-treatment and 2h/24h post-treatment using the VevoLAZR (21 MHz, 750/850 nm). The volumetric tumor sO2 was quantified using histogram distributions and the average mode was computed. The vascularization index (VI), a PD metric of tumor vessel density, was studied along with the sO2 mode by comparing changes at 2h with pre-treatment. Mice whose pre-treatment sO2 levels were over 65%, exhibited a 15% drop in oxygenation at 2h, remaining unchanged by 24h. Examining the sO2 and VI relationships revealed differences between the groups. All groups (except control) exhibited a positive correlation when the ∆VI was plotted as a function of ∆sO2 (r2≥0.85). Mice in the XRT/MB group had the largest slope (11.7) suggesting that a change in sO2 was accompanied by the largest change in vessel density. The slope of the USMB and XRT treatments was 5.6 and 2.9, respectively. The combination treatment induced the largest changes in vessel density and sO2. Early PA estimates of tumor oxygenation appear to correlate with the treatment-induced vascular changes. Such measure could potentially be used for predicting treatment outcome.

Spectral analysis of photoacoustic (PA) signals in the ultrasound frequency domain is a method that analyzes the power spectrum of PA signals to quantify tissue microstructures. PA spectral analysis has been correlated to changes in the size, morphology and concentration of absorbers that are smaller than the system spatial resolution. However, the calculated spectral parameters are still not system independent due to difficulty in eliminating variations in the light distribution for different optical wavelengths. Changes in spectral parameters for the same absorber geometry but different optical illumination wavelengths needs to be carefully examined. A gelatin vessel phantom is used. The vessels contain red blood cells comprised of oxy, deoxy and methemoglobin induced using oxygen, sodium hydrosulfite and sodium nitrite, respectively. The samples were imaged using the VevoLAZR system at wavelengths 680 – 905 nm in steps of 15 nm. The radiofrequency (RF) signals were analyzed to calculate the spectral slope. The results were compared to simulated RF signals acquired using the mcxyz Monte Carlo package coupled to the solution of the PA wave equation using the Green’s function approach. Changes in the spectral slope as a function of optical wavelength were detected. For longer optical wavelengths, the spectral slope increased for deoxyhemoglobin, but decreased for oxyhemoglobin and methemoglobin. The changes in the spectral slope were correlated to changes in the fluence distribution as optical properties change for different wavelengths. The change in the spectral slope as a function of optical wavelength and chromophore content can potentially be used in spectral unmixing for better estimation of hemoglobin content.

Red blood cell (RBC) transfusion is a critical component of the health care services. RBCs are stored in blood bags in hypothermic temperatures for a maximum of 6 weeks post donation. During this in vitro storage period, RBCs have been documented to undergo changes in structure and function due to mechanical and biochemical stress. Currently, there are no assessment methods that monitor the quality of RBCs within blood bags stored for transfusion. Conventional assessment methods require the extraction of samples, consequently voiding the sterility of the blood bags and potentially rendering them unfit for transfusions. It is hypothesized that photoacoustic (PA) technology can provide a rapid and non-invasive indication of RBC quality. In this study, a novel PA setup was developed for the acquisition of oxygen saturation (SO2) of two blood bags in situ. These measurements were taken throughout the lifespan of the blood bags (42 days) and compared against the clinical gold standard method of the blood gas analyzer (BGA). SO2 values of the blood bags increased monotonically throughout the storage period. A strong correlation between PA SO2 and BGA SO2 was found, however, PA values were on average 3.5% lower. Both techniques found the bags to increase by an SO2 of approximately 20%, and measured very similar rates of SO2 change. Future work will be focused on determining the cause of discrepancy between SO2 values acquired from PA versus BGA, as well as establishing links between the measured SO2 increase and other changes in RBC in situ.

We have developed a low-cost, non-contact, multispectral photoacoustic microscope system to study the functional parameters of cellular choromophores. The system uses low power continuous wave lasers and a photoacoustic sensor made of a kHz microphone coupled to a resonant chamber. Methemoglobin has relatively high optical absorption at 500 nm and 630 nm. Moreover, it has an almost the same optical absorption as hemoglobin at the isosbestic point of 525 nm. Photoacoustic data collected from methemoglobin using our system at wavelengths of 473 nm, 533 nm, and 633 nm show the similar trends as the methemoglobin optical absorption spectrum. The PA amplitude at 473 nm is about 1.03 times greater than at 533 nm and about 2.4 times greater than at 633 nm. Similarly, it possesses optical absorption of about 1.08 greater than at 533 nm and 1.34 times greater than at 633 nm. The developed system can be used as a differential photoacoustic microscope.

Photoacoustic (PA) field calculations using a Green’s function approach is presented. The method has been applied to predict PA spectra generated by normal (discocyte) and pathological (stomatocyte) red blood cells (RBCs). The contours of normal and pathological RBCs were generated by employing a popular parametric model and accordingly, fitted with the Legendre polynomial expansions for surface parametrization. The first frequency minimum of theoretical PA spectrum approximately appears at 607 MHz for a discocyte and 410 MHz for a stomatocyte when computed from the direction of symmetry axis. The same feature occurs nearly at 247 and 331 MHz, respectively, for those particles when measured along the perpendicular direction. The average experimental spectrum for normal RBCs is found to be flat over a bandwidth of 150-500 MHz when measured along the direction of symmetry axis. For spherical RBCs, both the theoretical and experimental spectra demonstrate negative slope over a bandwidth of 250-500 MHz. Using the Green’s function method discussed, it may be possible to rapidly characterize cellular morphology from single-particle PA spectra.

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 photo-acoustic imaging.

In the present paper, the optical wavelength dependence on the photoacoustic (PA) assessment of the pulsatile blood flow was investigated by means of the experimental and theoretical approaches analyzing PA radiofrequency spectral parameters such as the spectral slope (SS) and mid-band fit (MBF). For the experimental approach, the pulsatile flow of human whole blood at 60 bpm was imaged using the VevoLAZR system (40-MHz-linear-array probe, 700-900 nm illuminations). For the theoretical approach, a Monte Carlo simulation for the light transmit into a layered tissue phantom and a Green’s function based method for the PA wave generation was implemented for illumination wavelengths of 700, 750, 800, 850 and 900 nm. The SS and MBF for the experimental results were compared to theoretical ones as a function of the illumination wavelength. The MBF increased with the optical wavelength in both theory and experiments. This was expected because the MBF is representative of the PA magnitude, and the PA signal from red blood cell (RBC) is dependent on the molar extinction coefficient of oxyhemoglobin. On the other hand, the SS decreased with the wavelength, even though the RBC size (absorber size which is related to the SS) cannot depend on the illumination wavelength. This conflicting result can be interpreted by means of the changes of the fluence pattern for different illumination wavelengths. The SS decrease with the increasing illumination wavelength should be further investigated.

Imaging methods capable of indicating the potential for success of an individualized treatment course, during or immediately following the treatment, could improve therapeutic outcomes. Temperature Sensitive Liposomes (TSLs) provide an effective way to deliver chemotherapeutics to a localized tumoral area heated to mild-hyperthermia (HT). The high drug levels reached in the tumor vasculature lead to increased tumor regression via the cascade of events during and immediately following treatment. For a TSL carrying doxorubicin (DOX) these include the rapid and intense exposure of endothelial cells to high drug concentrations, hemorrhage, blood coagulation and vascular shutdown. In this study, ultrasound-guided photoacoustic imaging was used to probe the changes to tumors following treatment with the TSL, HaT-DOX (Heat activated cytoToxic). Levels of oxygen saturation (sO2) were studied in a longitudinal manner, from 30 min pre-treatment to 7 days post-treatment. The efficacious treatments of HT-HaT-DOX were shown to induce a significant drop in sO2 (10%) as early as 30 min post-treatment that led to tumor regression (in 90% of cases); HT-Saline and non-efficacious HT-HaT-DOX (10% of cases) treatments did not show any significant change in sO2 at these timepoints. The changes in sO2 were further corroborated with histological data, using the vascular and perfusion markers CD31 and FITC-lectin. These results allowed us to further surmise a plausible mechanism of the cellular events taking place in the TSL treated tumor regions over the first 24 hours post-treatment. The potential for using photoacoustic imaging to measure tumor sO2 as a surrogate prognostic marker for predicting therapeutic outcome with a TSL treatment is demonstrated.

Red blood cells (RBCs) stored in hypothermic environments for the purpose of transfusion have been documented to undergo structural and functional changes over time. One sign of the so-called RBC storage lesion is irreversible damage to the cell membrane. Consequently, RBCs undergo a morphological transformation from regular, deformable biconcave discocytes to rigid spheroechinocytes. The spherically shaped RBCs lack the deformability to efficiently enter microvasculature, thereby reducing the capacity of RBCs to oxygenate tissue. Blood banks currently rely on microscope techniques that include fixing, staining and cell counting in order to morphologically characterize RBC samples; these methods are labor intensive and highly subjective. This study presents a novel, high-throughput RBC morphology characterization technique using image flow cytometry (IFC). An image segmentation template was developed to process 100,000 images acquired from the IFC system and output the relative spheroechinocyte percentage. The technique was applied on samples extracted from two blood bags to monitor the morphological changes of the RBCs during in vitro hypothermic storage. The study found that, for a given sample of RBCs, the IFC method was twice as fast in data acquisition, and analyzed 250-350 times more RBCs than the conventional method. Over the lifespan of the blood bags, the mean spheroechinocyte population increased by 37%. Future work will focus on expanding the template to segregate RBC images into more subpopulations for the validation of the IFC method against conventional techniques; the expanded template will aid in establishing quantitative links between spheroechinocyte increase and other RBC storage lesion characteristics.

High-frequency ultrasound (>20 MHz) spectroscopy can be used to detect noninvasively DNA damage in cell samples in vitro, and in live tissue both ex vivo and in vivo. This chapter focuses on the former two aspects. Experimental evidence suggests that morphological changes that occur in cells undergoing apoptosis result in changes in frequency-dependent ultrasound backscatter. With advances in research, ultrasound spectroscopy is advancing the boundaries of fast, label-free, noninvasive DNA damage detection technology with potential use in personalized medicine and early therapy response monitoring. Depending on the desired resolution, parametric ultrasound images can be computed and displayed within minutes to hours after ultrasound examination for cell death.

In this chapter, we describe two new methodologies: (1) application of high-frequency ultrasound spectroscopy for in vivo detection of cancer cell death in small animal models, and (2) extension of ultrasound spectroscopy to the lower frequency range (i.e., 1-10 MHz range) for the detection of cell death in vivo in preclinical and clinical settings. Experiments using tumor xenografts in mice and cancer treatments based on chemotherapy are described. Finally, we describe how one can detect cancer response to treatment in patients noninvasively early (within 1 week of treatment initiation) using low-frequency ultrasound spectroscopic imaging and advanced machine learning techniques. Color-coded images of ultrasound spectroscopic parameters, or parametric images, permit the delineation of areas of dead cells versus viable cells using high ultrasound frequencies, and the delineation of areas of therapy response in patient tumors using clinically relevant ultrasound frequencies. Depending on the desired resolution, parametric ultrasound images can be computed and displayed within minutes to hours after ultrasound examination for cell death. A noninvasive and express method of cancer response detection using ultrasound spectroscopy provides a framework for personalized medicine with regards to the treatment planning of refractory patients resulting in substantial improvements in patient survival.

Poly(lactide-co-glycolic acid) (PLGA) particles are biocompatible and bio­degradable, and can be used as a carrier for various chemotherapeutic drugs, imaging agents and targeting moieties. Micrometer-sized PLGA particles were synthesized with gold nanoparticles and DiI dye within the PLGA shell, and perfluorohexane liquid (PFH) in the core. Upon laser irradiation, the PLGA shell absorbs the laser energy, activating the liquid core (liquid conversion to gas). The rapidly expanding gas is expelled from the particle, resulting in a microbubble; this violent process can cause damage to cells and tissue. Studies using cell cultures show that PLGA particles phagocytosed by single cells are consistently vaporized by laser energies of 90 mJ cm−2, resulting in cell destruction. Rabbits with metastasized squamous carcinoma in the lymph nodes are then used to evaluate the anti-cancer effects of these particles in the lymph nodes. After percutaneous injection of the particles and upon laser irradiation, through the process of optical droplet vaporization, ultrasound imaging shows a significant increase in contrast in comparison to the control. Histology and electron microscopy confirm damage with disrupted cells throughout the lymph nodes, which slows the tumor growth rate. This study shows that PLGA particles containing PFC liquids can be used as theranostic agents in vivo.

// Ali Sadeghi-Naini 1,2,3,4 , Lakshmanan Sannachi 1,2,3,4 , Kathleen Pritchard 5 , Maureen Trudeau 5 , Sonal Gandhi 5 , Frances C. Wright 6,7 , Judit Zubovits 8 , Martin J. Yaffe 1,3 , Michael C. Kolios 3,9 , Gregory J. Czarnota 1,2,3,4 1 Imaging Research - Physical Sciences, Sunnybrook Research Institute, Sunnybrook Health Sciences Centre, Toronto, ON, Canada 2 Department of Radiation Oncology, Odette Cancer Centre, Sunnybrook Health Sciences Centre, Toronto, ON, Canada 3 Department of Medical Biophysics, Faculty of Medicine, University of Toronto, Toronto, ON, Canada 4 Department of Radiation Oncology, Faculty of Medicine, University of Toronto, Toronto, ON, Canada 5 Department of Medical Oncology, Sunnybrook Health Sciences Centre, and Faculty of Medicine, University of Toronto, Toronto, ON, Canada 6 Division of General Surgery, Sunnybrook Health Sciences Centre, Toronto, ON, Canada 7 Department of Surgery, Faculty of Medicine, University of Toronto, Toronto, ON, Canada 8 Department of Pathology, Sunnybrook Health Sciences Centre, and Faculty of Medicine, University of Toronto, Toronto, ON, Canada 9 Department of Physics, Ryerson University, Toronto, ON, Canada Correspondence: Gregory J. Czarnota, email: // Keywords : Personalized medicine, Therapy response monitoring, Breast Cancer, Neo-adjuvant chemotherapy, Tumor response heterogeneity, Textural analysis, Quantitative ultrasound. Received : March 12, 2014 Accepted : May 6, 2014 Published : May 7, 2014 Abstract Early alterations in textural characteristics of quantitative ultrasound spectral parametric maps, in conjunction with changes in their mean values, are demonstrated here, for the first time, to be capable of predicting ultimate clinical/pathologic responses of breast cancer patients to chemotherapy. Mechanisms of cell death, induced by chemotherapy within tumor, introduce morphological alterations in cancerous cells, resulting in measurable changes in tissue echogenicity. We have demonstrated that the development of such changes is reflected in early alterations in textural characteristics of quantitative ultrasound spectral parametric maps, followed by consequent changes in their mean values. The spectral/textural biomarkers derived on this basis have been demonstrated as non-invasive surrogates of breast cancer chemotherapy response. Particularly, spectral biomarkers sensitive to the size and concentration of acoustic scatterers could predict treatment response of patients with up to 80% of sensitivity and specificity (p=0.050), after one week within 3-4 months of chemotherapy. However, textural biomarkers characterizing heterogeneities in distribution of acoustic scatterers, could differentiate between treatment responding and non-responding patients with up to 100% sensitivity and 93% specificity (p=0.002). Such early prediction permits offering effective alternatives to standard treatment, or switching to a salvage therapy, for refractory patients.

31 ISSN 1758-1923 10.2217/BMT.13.58 © 2014 Future Medicine Ltd Breast Cancer Manage. (2014) 3(1), 31–35 Despite the realization that breast cancer is composed of many subtypes with unique molecular signatures that are related to chemotherapy response, the majority of chemotherapy administered is standardized, with most patients receiving anthracyclineand taxanebased treatments. For upfront chemotherapy, an opportunity exists to monitor and customize treatments adaptively, as tumors in situ can serve as a response biomarker. Noninvasive functional imaging methods that can detect early tumor response can thus begin to be used to adaptively change chemotherapy. Such tests could facilitate switches from ineffective treatments early on during chemotherapy (days to weeks), as opposed to patients receiving months of a treatment to which tumor cells do not respond. Quantitative ultrasound (QUS) is one such method that is sensitive to cell death and is being investigated as a functional breast cancer chemotherapy response imaging method. Breast cancer chemotherapy & response monitoring Oncology, as a clinical field, involves classifying tumor stages and subtypes based on anatomical and pathological findings, which includes histological examination of tumor samples from individual patients, for example HER2/neu in breast cancer. This involves identifying markers associated with prognosis, which often, but not always, portend treatment response. The history of cancer therapy suggests that there is no optimized solution for all patients and, hence, cancer treatments include the use of chemotherapy drugs, ionizing radiation, immunotherapy, gene therapy, surgery or a combination of some of these treatments. However, there are variations in tumor response to a given treatment from one patient to another due to the differences in complexity in individual tumor biology. Personalized medicine is proposed as the customization of clinical therapy based on a patient’s tumor biology. Therapy response monitoring is one of the important parts

Solid tumors are typically supplied nutrients by a network of irregular blood vessels. By targeting these vascular networks, it might be possible to hinder cancer growth and metastasis. Vascular disrupting agents induce intertumoral hemorrhaging, making photoacoustic (PA) imaging well positioned to detect bleeding due to its sensitivity to hemoglobin and its various states. We introduce a fractal-based numerical model of intertumoral hemorrhaging to simulate the PA signals from disrupted tumor blood vessels. The fractal model uses bifurcated cylinders to represent vascular trees. To mimic bleeding from blood vessels, hemoglobin diffusion from microvessels was simulated. In the simulations, the PA signals were detected by a linear array transducer (30 MHz center frequency) of four different vascular trees. The power spectrum of each beamformed PA signal was computed and fitted to a straight line within the −6-dB bandwidth of the receiving transducer. The spectral slope and midband fit (MBF) based on the fit decreased by 0.11 dB / MHz and 2.12 dB, respectively, 1 h post bleeding, while the y-intercept increased by 1.21 dB. The results suggest that spectral PA analysis can be used to measure changes in the concentration and spatial distribution of hemoglobin in tissue without the need to resolve individual vessels. The simulations support the feasibility of using PA imaging and spectral analysis in cancer treatment monitoring by detecting microvessel disruption.

Phase change nanoemulsions have been proposed as theranostic agents, using light to induce vaporization into bubbles (also called optical droplet vaporization). The current work uses perfluorohexane nanoemulsions (PFH-NEs) stabilized by a highly biocompatible and optically absorbing fluorosurfactant shell. Once vaporized, the bubbles can be used for contrast enhanced ultrasound (CEUS) imaging but also to enhance photoacoustic (PA) signals due to the presence of bubbles and optical absorbing shell material. The formation and expansion of these gas filled bubbles leads to increasing photoacoustic signals for imaging. Compared to other contrast agents which may not give stable signals due to photo-degradation, these contrast agents are shown to be stable up to 24 hours. The source of PA signal enhancement is through the presence of long lasting perfluorohexane (PFH) bubbles resulting from the optical vaporization. These bubbles generated from the PFH-NEs directly generate photoacoustic signals due to the optical absorption from the fluorosurfactant shell, but also secondary signals from the subsequent scattering of the photoacoustic waves from the PFH bubbles. In addition, the pressures generated from vaporization of NEs and ability to load chemotherapeutic agents enable these nanoparticles to also be used for cancer therapy by contributing to drug delivery and transport.

We have developed a photoacoustic (PA) sensor using a low-power, continuous- wave laser and a kHz-range microphone. The sensor is simple, flexible, cost-effective, and compatible with commercial optical microscopes. The sensor enables noncontact PA measurements through air, whereas most current existing PA techniques require an acoustic coupling liquid for detection. The PA sensor has three main components: one is the chamber that holds the sample, the second is a resonator column used to amplify the weak PA signals generated within the sample chamber, and the third is a microphone at the end of the resonator column to detect the amplified signals. The chamber size was designed to be 8 mm × 3 mm as the thermal diffusion length and viscous-thermal damping of air at room pressure and temperature are 2 and 1 mm, respectively. We numerically and experimentally examined the effect of the resonator column size on the frequency response of the PA sensor. The quality factor decreased significantly when the sample chamber size was reduced from 4 mm × 3 mm to 2 mm × 3 mm due to thermos-viscous damping of the air. The quality factor decreased by 27%, demonstrating the need for optimal design for the sample chamber and resonator column size. The system exhibited noise equivalent molecular sensitivity (NEM) per unit bandwidth (NEM / √ Δ f ) of ∼19,966 Hz − 1/2 or 33 × 10 − 21 mol or 33 zeptomol, which is an improvement of 2.2 times compared to the previous system design. This PA sensor has the potential for noncontact high-resolution PA imaging of materials without the need for coupling fluids.

Nanoemulsions serve as promising biomedical agents for cancer therapy and imaging. Recently, the development of perfluorocarbon (PFC) nanoemulsions has led to the ability of these nanoparticles to encapsulate therapeutic agents and vaporize into microbubbles and nanobubbles leading to cancer cell death. The PFC bubbles formed from nanoemulsions can serve as ultrasound contrast agents that can be used to verify the delivery of the nanoemulsion payload. In this work, perfluorohexane nanoemulsions (PFH-NEs) were synthesized for use as theranostic agents, by both encapsulating these nanoparticles with different chemotherapeutic agents (i.e., paclitaxel, doxorubicin) as well as to use the laser induced PFH bubbles to enhance the nonlinear ultrasound signals from cells for treatment monitoring. Since nonlinear ultrasound contrast arises specifically from the nonlinear scattering from oscillating microbubbles and nanobubbles and does not account for linear scattering from tissue, these PFH-NEs have the potential to effectively locate the tumor region for treatment monitoring.

Lipid shell-stabilized nanobubbles (NB

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

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

Evidence suggests that compression and shear wave elastography are sensitive to the mechanical property changes occuring in dying cells following chemotherapy, and can hence be used to monitor cancer treatment response. A qualitative and quantitative understanding of the mechanical changes at the cellular level would allow to better infer how these changes affect macroscopic tissue mechanical properties and therefore allow the optimization of elastographic techniques (such as shear wave elastography) for the monitoring of cancer therapy. We used intracellular particle tracking microrheology (PTM) to investigate the mechanical property changes of cells exposed to paclitaxol, a mitotic inhibitor used in cancer chemotherapy. The average elastic and viscous moduli of the cytoplasm of treated MCF-7 breast cancer cells were calculated for frequency ranges between 0.2 and 100 rad s–1 (corresponding to 0.03 and 15.92 Hz, respectively). A significant increase in the complex shear modulus of the cell cytoplasm was detected at 12 h post treatment. At 24 h after drug exposure, the elastic and viscous moduli increased by a total of 191.3 Pa (>8000×) and 9 Pa (~9×), respectively for low frequency shear modulus measurements (at 1 rad s–1). At higher frequencies (10 rad s–1), the elastic and viscous moduli increased by 188.5 Pa (~60×) and 1.7 Pa (~1.1×), respectively. Our work demonstrates that PTM can be used to measure changes in the mechanical properties of treated cells and that cell elasticity significantly increases by 24 h after chemotherapy exposure.

Cancer is a major public health problem all over the world. It is currently the second leading cause of death in the United States, and is expected to surpass heart diseases as the leading cause of death in the next few years. To overcome this problem, researchers are discovering new treatments and developing imaging technologies that can aid in early detection of cancer. In this work, we investigate the use of signal envelope statistics to monitor and quantify structural changes in tissues during cell death. Ultrasound backscatter and photoacoustic data were obtained from three mice treated with, Hat-DOX, DOX and saline. The signal envelope statistics were examined by fitting the Rayleigh and Generalized Gamma distributions. The fit parameters showed sensitivity to structural changes in the cells in photoacoustic and ultrasound images. The results indicate that photoacoustic signal statistics can be potentially used to monitor structural changes within a tumor, raising the possibility of monitoring cancer therapy efficacy.

The nucleus-to-cytoplasmic (N:C) ratio of a cell is often used when assessing histology for the presence of malignant disease. In this proof of concept study, we present a new, non-optical method for determination of the N:C ratio using ultra-high Frequency ultrasound (US) and photoacoustics (PA). When using transducers in the 100 MHz–500 MHz range, backscattered US pulses and emitted PA waves are encoded with information pertaining to the dimension and morphology of micron-sized objects. If biological cells are interrogated, the diameter of the scattering or absorbing structure can be assessed by fitting the power spectra of the measured US or PA signals to theoretical models for US backscatter and PA emission from a fluid sphere. In this study, the cell and nucleus diameters of 9 MCF-7 breast cancer cells were determined using a new simplified model that calculates the theoretical values of the location of the power spectra minima for both US and PA signals. These diameters were then used to calculate the N:C ratio of the measured cells. The average cell diameter determined by US pulses from a transducer with a central frequency of 375 MHz was found to be $$15.5\,\upmu \hbox {m}\pm \,1.8\,\upmu \hbox {m}$$ . The PA waves emitted by the cell nuclei were used to determine an average nuclear diameter of $$12.0\,\upmu \hbox {m}\pm 1.3\,\upmu \hbox {m}$$ . The N:C ratio for these cells was calculated to be $$1.9\pm 1.0$$ , which agrees well with previously reported N:C values for this cell type.