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In this work we demonstrate the potential of using nanobubbles (NBs) as radiation therapy enhancers for maximizing tumoral cell death. Photoacoustic (PA) imaging can be used to probe the mechanism of action of this treatment due to its ability to examine the oxygenation of tumors. In vivo experiments were performed in mice bearing prostate cancer tumors and the NB therapies were compared with conventional microbubble (MB) treatments combined with radiation. Our preliminary results show that NB combined with a single dose of 8 Gy radiation induce 40% tumor cell death compared to 20% observed with MB treatments. PA imaging suggest that NBs have an extravascular effect.

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.

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

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.

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

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.

Nanobubbles are a new class of ultrasound contrast agents. Unlike conventional microbubbles, their sub-micron (~200nm) diameter allows them to extravasate outside the vasculature and accumulate in the tumor interstitium. In this study, nanobubbles with shells loaded with Sudan Black (BNB) and DiD fluorescent dye were synthesized. These nanobubbles can be used to simultaneously enhance ultrasound and photoacoustic signals for in vivo breast tumor imaging. The nanobubbles consisted of lipid shells with a C3F8 gas core and were formed via self-assembly driven by mechanical agitation and size isolation via centrifugation. Herceptin antibody was conjugated to the BNB for targeting HER2-positive cells via standard EDC/NHS coupling chemistry. Human breast cancer cell lines (BT474 as HER2-positive and MDA-MB-231 as HER2-negative) were inoculated in the flanks of BALB/c-B17-Scid mice. Ultrasound and photoacoustic imaging (VevoLAZR, 21MHz, 720nm) were performed pre-injection and post-injection of the Herceptin conjugated BNB. The impact of Herceptin targeting was assessed by computing the PA frequency spectra and the non-linear contrast US images of the tumor regions. Photoacoustic images of the HER2-positive tumor showed an average of 6 dB increase in contrast signal 2 mins post-injection, while the HER2-negative MDA tumors showed a negligible change in image contrast, suggesting increased uptake of Herceptin labelled BNBs. The enhanced contrast is also confirmed by the non-linear contrast signals between positive and negative tumors. The photoacoustic technique can potentially be used to examine the kinetics of BNB extravasation. This work shows the potential of BNBs as multi-modal contrast agents capable of specialized tumor imaging in vivo.

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.

Optical coherence tomography (OCT) is used to provide anatomical information of biological systems but can also provide functional information by characterizing the motion of intracellular structures. Dynamic light scattering OCT was performed on intact, control MCF-7 breast cancer cells and cells either treated with paclitaxel to induce apoptosis or deprived of nutrients to induce oncosis. Autocorrelations (ACs) of the temporal fluctuations of OCT intensity signals demonstrate a significant decrease in decorrelation time after 24 h in both the paclitaxel-treated and nutrient-deprived cell groups but no significant differences between the two groups. The acquired ACs were then used as input for the CONTIN deconvolution algorithm, and all produced CONTIN outputs with three distinct peaks for all experimental conditions. After 24 h of either paclitaxel treatment or nutrient deprivation, the area-under-the-curve (AUC) of the first peak increased significantly while the AUC of the third peak decreased significantly. These results lend strong support to the hypothesis that ACs acquired from cells are composed of multiple components that correspond to light scattered by different subcellular structures and organelles.

During the in vitro storage of red blood cells (RBCs), unfavourable changes (storage lesions) cause a rapid consumption of intracellular diphosphoglycerate. The latter deregulates the oxygen-haemoglobin binding potential, subsequently increasing oxygen saturation (SOA photoacoustic (PA) imaging technique was developed for acquiring the SOA strong correlation (rThis study suggests that PA imaging can non-invasively track the SO

A resonant mass measurement technique simultaneously distinguishes and characterizes (size and concentration) buoyant and non-buoyant particles in a bubble sample., Nano-sized shell-stabilized gas bubbles have applications in various fields ranging from environmental science to biomedical engineering. A resonant mass measurement (RMM) technique is demonstrated here as a new and only method capable of simultaneously measuring the size and concentration of buoyant and non-buoyant particles in a nanobubble sample used as a next-generation ultrasound contrast agent.

The nucleus-to-cytoplasm ratio (N:C) can be used as one metric in histology for grading certain types of tumor malignancy. Current N:C assessment techniques are time-consuming and low throughput. Thus, in high-throughput clinical contexts, there is a need for a technique that can assess cell malignancy rapidly. In this study, we assess the N:C ratio of four different malignant cell lines (OCI-AML-5—blood cancer, CAKI-2—kidney cancer, HT-29— colon cancer, SK-BR-3—breast cancer) and a non-malignant cell line (MCF-10A –breast epithelium) using an imaging flow cytometer (IFC). Cells were stained with the DRAQ-5 nuclear dye to stain the cell nucleus. 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 5284 OCI-AML-5 cells, 1096 CAKI-2 cells, 6302 HT-29 cells, 3159 SK-BR-3 cells, and 1109 MCF-10A cells. The N:C ratio was calculated as the ratio of the nucleus diameter to the total cell diameter. The average cell and nucleus diameters from IFC were 12.3±1.2μm and 9.0±1.1μm for OCI-AML5 cells, 24.5±2.6μm and 15.6±2.1μm for CAKI-2 cells, 16.2±1.8μm and 11.2±1.3μm for HT29 cells, 18.0±3.7μm and 12.5±2.1μm for SK-BR-3 cells, and 19.4±2.2μm and 10.1± 1.8μm for MCF-10A cells. Here we show a general N:C ratio of ~0.6–0.7 across varying malignant cell lines and a N:C ratio of ~0.5 for a non-malignant cell line. This study demonstrates the use of IFC to assess the N:C ratio of cancerous and non-cancerous cells, and the promise of its use in clinically relevant high-throughput detection scenarios to supplement current workflows used for cancer cell grading.

The low delivery efficiency of nanoparticles to solid tumors greatly reduces the therapeutic efficacy and safety which is closely related to low permeability and poor distribution at tumor sites. In this work, an “intrinsic plus extrinsic superiority” administration strategy is proposed to dramatically enhance the mean delivery efficiency of nanoparticles in prostate cancer to 6.84% of injected dose, compared to 1.42% as the maximum in prostate cancer in the previously reported study. Specifically, the intrinsic superiority refers to the virus‐mimic surface topology of the nanoparticles for enhanced nano–bio interactions. Meanwhile, the extrinsic stimuli of microbubble‐assisted low‐frequency ultrasound is to enhance permeability of biological barriers and improve intratumor distribution. The enhanced intratumor enrichment can be verified by photoacoustic resonance imaging, fluorescence imaging, and magnetic resonance imaging in this multifunctional nanoplatform, which also facilitates excellent anticancer effect of photothermal treatment, photodynamic treatment, and sonodynamic treatment via combined laser and ultrasound irradiation. This study confirms the significant advance in nanoparticle accumulation in multiple tumor models, which provides an innovative delivery paradigm to improve intratumor accumulation of nanotherapeutics., This work proposes the combination of intrinsic optimization the nanostructure as virus‐mimic surface topology to enhance nano–bio interactions and extrinsic stimuli of microbubble‐assisted low‐frequency ultrasound to vastly improve intratumor permeability and distribution of nanoparticles in multiple cancer models. The multifunctional nanoplatform is capable with trimodal imaging to determine the optimal therapeutic timing, which demonstrates a successful combined anticancer effect.

Over the last two and a half decades, photoacoustic (PA) imaging has become an important area of research in biomedical optics. Combining the high contrast of optical imaging with the high spatial resolution of ultrasound (US) imaging, PA imaging can simultaneously visualize anatomical structures while interrogating their functionality through multiwavelength optical spectroscopy. Alongside technological developments and imaging applications in optical and acoustic resolution PA imaging, a family of PA signal analysis techniques can extract additional information about the sample being imaged. This Tutorial focuses on techniques that rely on the analysis of PA signals in a manner similar to that in the complimentary field of quantitative ultrasound (QUS) imaging of soft tissues. In QUS, signal analysis techniques have been developed to analyze the US signals resulting from the scattering of many unresolved scatterers within the resolution volume of the imaging device. The implementation of these US techniques in PA can enable new applications in biomedicine beyond traditional anatomical PA imaging, further increasing the utilization and impact of this promising modality.

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.

Phase-transition nanoparticles have been identified as effective theragnostic, anti-cancer agents. However, non-selective delivery of these agents results in inaccurate diagnosis and insufficient treatment. In this study, we report on the development of targeted phase-transition polymeric nanoparticles (NPs) for the imaging and treatment of breast cancer cell lines over-expressing human epidermal growth factor receptor 2 (HER2). These NPs contain a perfluorohexane liquid interior and gold nanorods (GNRs) stabilized by biodegradable and biocompatible copolymer PLGA-PEG. Water-insoluble therapeutic drug Paclitaxel (PAC) and fluorescent dye were encapsulated into the PLGA shell. The NP surfaces were conjugated to HER2-binding agent, Herceptin, to actively target HER2-positive cancer cells. We evaluated the potential of using these NPs as a photoacoustic contrast agent. The efficacy of cancer cell treatment by laser-induced vaporization and stimulated drug release were also investigated. The results showed that our synthesized PLGA-PEG-GNRs (mean diameter 285 ± 29 nm) actively targeted HER2 positive cells with high efficacy. The laser-induced vaporization caused more damage to the targeted cells versus PAC-only and negative controls. This agent may provide better diagnostic imaging and therapeutic potential than current methods for treating HER2-positive breast cancer.

Acoustic waves are dissipated when they pass through bubbly media. Dissipation by bubbles takes place through thermal damping (Td), radiation damping (Rd), and damping due to the friction of the liquid (Ld) and friction of the coating (Cd). Knowledge of the contributions of Td, Rd, Ld, and Cd during nonlinear bubble oscillations will help in optimizing bubble and ultrasound exposure parameters for the relevant applications by maximizing a desirable outcome or oscillation pattern. In this work, we investigate the mechanisms of dissipation in bubble oscillations and their contribution to the total damping (Wtotal) in various nonlinear regimes. By using a bifurcation analysis, we have classified nonlinear dynamics of bubbles that are sonicated with their third superharmonic (SuH) and second SuH resonance frequency (fr), pressure dependent resonance frequency (PDfr), fr, subharmonic (SH) resonance (fsh = 2fr), pressure dependent SH resonance (PDfsh), and 1/3 order SH resonance, which are important exposure ranges for various applications. The corresponding Td, Rd, Ld, Cd, Wtotal, scattering to dissipation ratio, maximum wall velocity, and maximum backscattered pressure from non-destructive oscillations of bubbles were calculated and analyzed using the bifurcation diagrams. Universal ultrasound exposure parameter ranges are revealed in which a particular non-destructive bubble related phenomenon (e.g., wall velocity) is enhanced. The enhanced bubble activity is then linked to relevant ultrasound applications. This paper represents the first comprehensive analysis of the nonlinear oscillations regimes, the corresponding damping mechanisms, and the bubble related phenomena.

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

Photoacoustic imaging has experienced exponential growth over the past decade, with many applications in biomedicine. One application ideally suited to the analysis of single cells is photoacoustic microscopy (PAM). Using PAM, detailed submicrometer resolution images of single cells can be produced, with contrast dependent primarily on the optical absorption properties of the cell. A multiwavelength approach for targeting specific endogenous or exogenous chromophores can enhance cellular detail and resolve single organelles with contrast not possible with traditional optical microscopy. A quantitative analysis of the photoacoustic signals acquired from single cells can provide insight into their anatomical, biomechanical, and functional properties. This information can be used to identify specific cells, or to enhance the understanding of biological processes at the single cell level. This comprehensive review on PAM covers recent advances in high-resolution PAM, signal processing methods, and potential clinical applications targeting single cells in vitro and in vivo .

Non-invasive monitoring of cancer cell death would permit rapid feedback on treatment response. One technique showing such promise is quantitative ultrasound. High-frequency ultrasound spectral radiofrequency analysis was used to study cell death in breast cancer cell samples. Quantitative ultrasound parameters, including attenuation, spectral slope, spectral 0-MHz-intercept, midband fit, and fitted parameters displayed significant changes with paclitaxel-induced cell death, corresponding to observations of morphological changes seen in histology and electron microscopy. In particular, a decrease in spectral slope from 0.24±0.07 dB/MHz to 0.04±0.09 dB/MHz occurred over 24 hours of treatment time and was identified as an ultrasound parameter capable of differentiating post-mitotic arrest cell death from classical apoptosis. The formation of condensed chromatin aggregates of 1 micron or greater in size increased the number of intracellular scatterers, consistent with a hypothesis that nuclear material is a primary source of ultrasound scattering in dying cells. It was demonstrated that the midband fit quantitatively correlated to cell death index, with a Pearson R-squared value of 0.99 at p

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

This study presents the fundamental equations governing the pressure dependent disipation mechanisms in the oscillations of coated bubbles. A simple generalized model (GM) for coated bubbles accounting for the effect of compressibility of the liquid is presented. The GM was then coupled with nonlinear ODEs that account for the thermal effects. Starting with mass and momentum conservation equations for a bubbly liquid and using the GM, nonlinear pressure dependent terms were derived for power dissipation due to thermal damping (Td), radiation damping (Rd) and dissipation due to the viscosity of liquid (Ld) and coating (Cd). The pressure dependence of the dissipation mechanisms of the coated bubble have been analyzed. The dissipated energies were solved for uncoated and coated 2–20 μ m in bubbles over a frequency range of 0.25 f r - 2.5 f r ( f r is the bubble resonance) and for various acoustic pressures (1 kPa-300 kPa). Thermal effects were examined for air and C3F8 gas cores. In the case of air bubbles, as pressure increases, the linear thermal model looses accuracy and accurate modeling requires inclusion of the full thermal model. However, for coated C3F8 bubbles of diameter 1–8 μ m , which are typically used in medical ultrasound, thermal effects maybe neglected even at higher pressures. For uncoated bubbles, when pressure increases, the contributions of Rd grow faster and become the dominant damping mechanism for pressure dependent resonance frequencies (e.g. fundamental and super harmonic resonances). For coated bubbles, Cd is the strongest damping mechanism. As pressure increases, Rd contributes more to damping compared to Ld and Td. For coated bubbles, the often neglected compressibility of the liquid has a strong effect on the oscillations and should be incorporated in models. We show that the scattering to damping ratio (STDR), a measure of the effectiveness of the bubble as contrast agent, is pressure dependent and can be maximized for specific frequency ranges and pressures.

Photoacoustic (PA) signals encode information about the optical absorption and spatial distribution of absorbing chromophores as well as the light distribution in the medium. The wavelength dependence of the latter affects the accuracy in chromophore quantification, including estimations of oxygen saturation (sO2) with depth. We propose the use of the ratio of the PA radiofrequency (RF) spectral slopes (SS) at different optical wavelengths to generate frequency filters which can be used to match the fluence profiles across separate images generated with different optical wavelengths. Proof-of-principle experiments were carried on a plastic tube with blood of a known oxygenation inserted into a porcine tissue. The algorithm was tested in-vivo in the hind leg of six CD1 mice, each under three different breathing conditions (100 % O2, room air and 100 % CO2). Imaging was done using the VevoLAZR system at the wavelengths 720 and 870 nm. The SS was calculated from the linear fit of the ratio of the photoacoustic RF power spectra at the two wavelengths. An ultrasound frequency filter was designed and applied to each segmented PA signal in the frequency domain and inversely transformed into the time domain to correct for the differences in the fluence profiles at both wavelengths. Linear spectral unmixing was used to estimate sO2 before and after applying the ultrasound frequency filter. The estimated blood sO2 in the plastic tube for the porcine tissue experiment improved by 10.3% after applying the frequency filter when compared to the sO2 measured by a blood gas analyzer. For the in-vivo mouse experiments, the applied sO2 correction was 2.67, 1.33 and -3.33% for every mm of muscle tissue for mice breathing 100% O2, room air and 100% CO2, respectively. The approach presented here provides a new approach for fluence matching that can potentially improve the accuracy of sO2 estimates by removing the fluence depth dependence at different optical wavelengths.

In this work, through applying a comprehensive bifurcation analysis method, we study the nonlinear radial oscillations of the bubble oscillator. The frequency of the driving force is chosen as the linear resonance frequency ( f r ) and linear subharmonic (SH) resonance frequency ( f s h = 2 f r ) of the bubble. Results show that, when the bubble is sonicated with 2 f r , period doubling is more likely to result in non-destructive oscillations. The evolution of the period-2 (P2) oscillations of the bubble makes the shape of a bowtie for bubbles with an initial diameter of 0.74 μm and above. When f = 2 f r , the phase portrait of the P2 attractor is distinctly different from a P2 attractor when f = f r , and subharmonic component of the backscattered pressure is maximum. When sonicated by 2 f r , due to lower oscillation amplitude and gentler bubble collapse, the bubble can sustain stable P2 oscillations for a longer duration and over a broader range of applied acoustic pressure.

BackgroundNeoadjuvant chemotherapy (NAC) is the standard of care for patients with locally advanced breast cancer (LABC). The study was conducted to investigate the utility of quantitative ultrasound (QUS) carried out during NAC to predict the final tumour response in a multi-institutional setting.MethodsFifty-nine patients with LABC were enrolled from three institutions in North America (Sunnybrook Health Sciences Centre (Toronto, Canada), MD Anderson Cancer Centre (Texas, USA), and Princess Margaret Cancer Centre (Toronto, Canada)). QUS data were collected before starting NAC and subsequently at weeks 1 and 4 during chemotherapy. Spectral tumour parametric maps were generated, and textural features determined using grey-level co-occurrence matrices. Patients were divided into two groups based on their pathological outcomes following surgery: responders and non-responders. Machine learning algorithms using Fisher's linear discriminant (FLD), K-nearest neighbour (K-NN), and support vector machine (SVM-RBF) were used to generate response classification models.ResultsThirty-six patients were classified as responders and twenty-three as non-responders. Among all the models, SVM-RBF had the highest accuracy of 81% at both weeks 1 and week 4 with area under curve (AUC) values of 0.87 each. The inclusion of week 1 and 4 features led to an improvement of the classifier models, with the accuracy and AUC from baseline features only being 76% and 0.68, respectively.ConclusionQUS data obtained during NAC reflect the ongoing treatment-related changes during chemotherapy and can lead to better classifier performances in predicting the ultimate pathologic response to treatment compared to baseline features alone.

Permeabilization of the cell membrane can be achieved through techniques such as sonoporation and microinjection. An alternative method is optoporation that uses tightly focused laser pulses typically from femtosecond lasers. Localized damage/modification of the plasma membrane has been shown using light absorbing nanoparticles heated with short laser pulses. In this work, we use targeted nanobubbles stained with Sudan Black B for purposes of enhancing optoporation without using femtosecond lasers as well as a contrast agent for photoacoustic microscopy. Nanobubbles were targeted with antibody Herceptin and added to each petri dish before being incubated for 4 hours before photoacoustic imaging. Fluorescence microscopy was used to confirm presence of the nanobubbles using DiO lipid stain. The photoacoustic microscope is equipped with a 400MHz transducer and 532nm laser focused through a 10x objective. The focal spots of laser and transducer were co-aligned and a portion of the leaser beam was directed to a joulemeter for energy measurement. Propidium Iodide was used to detect poration where uptake was not seen in healthy cells but uptake was seen as early as 7 minutes in nanobubble loaded cells. PA signals were detected from cells loaded with NBs and were not from the control group.

Photoacoustic (PA) imaging of blood flow can provide label free and non-invasive assessment of red blood cell (RBC) aggregation and the oxygen saturation (sO 2 ). Our group has previously demonstrated that the interrelationship between RBC aggregation and the sO 2 during a pulsatile blood flow could be potentially assessed using PA imaging. The pulsatile blood flow yields spatiotemporal changes in RBC aggregation, affecting PA imaging. A simple particle motion model was developed based on the blood flow velocity measured from the human radial artery (RA). The positions of randomly distributed, identical circular particles (2.7 um radius) in the lateral-axial plane (20 mm by 2 mm) were traced at each time step of an experimentally measured velocity profile. At each step, the time dependent PA power $(P_{PA})$ from each single cell (or particles interacting to form aggregates) was computed by modeling and accounting for the directivity of a 21 MHz (9.2 to 32.8 MHz bandwidth) linear array. In-vivo PA images of the RA of healthy volunteers were acquired using the VevoLAZR equipped with a 21 MHz linear-array probe. The measured PA images were compared to the simulated PA images. The aggregates formed a parabolic front along the axial direction and were driven to the right-hand side along the lateral direction as the simulation propagated in time. The $P_{PA}$ was also large at the parabolic front, and was also driven to the right-hand side for every time step. The spatiotemporal distribution of the computed $P_{PA}$ was comparable to the experimental $P_{PA}$. Specifically, the $P_{PA}$ increased by 12 dB along the lateral direction. These results can be used to study the label-free, non-invasive assessment of the spatiotemporal distribution of sO 2 in vivo. Furthermore, the improved particle model can provide insights into the mechanism of PA wave generation from RBC aggregation during in vivo blood flow.

Superparamagnetic iron-oxide nanoparticles (NP) have been designed for dual modal applications include photoacoustic (PA) imaging and photothermal therapy. As the approach efficacy depends on the cellular uptake, virus-shaped nanoparticles that could potentially increase particle uptake were developed. Laser illumination could be used for both PA imaging and photo-thermally treatment of cancer. Virus-like silica encapsulated superparamagnetic iron oxide NPs were developed with a rugged surface/nanoscaffold. Nanoparticle uptake was measured in PC-3 prostate cancer cell line and NP cell uptake as a function of time and concentration were investigated. The potential application in PA imaging and photothermal therapy was shown in in-vitro experiments.

We have developed an acoustic flow cytometer that combines ultrasound and microfluidics to rapidly size and count cells label-free in a high-throughput manner. Cells are hydrodynamically focused single file into a narrow stream through an ultrasound beam where ultrasound is scattered from each individual cell. The ultrasound operates at a center frequency of 375 MHz with a wavelength of 4 μm; in this regime, the power spectra of the backscattered ultrasound waves have distinct features at specific frequencies that are directly related to the cell size. Our approach can be used to determine the cell diameter by comparing these unique spectral features to established analytical solutions of ultrasound scattering. 3 μm beads with known scattering properties were used to validate the technique. Then, we examined two cells lines with different average cell diameters; acute myeloid leukemia cells, where 2,390 measurements resulted in a mean diameter of 10.0 ± 1.7 μm, and HT29 colorectal cancer cells, where 1,955 measurements resulted in a mean diameter of 15.0 ± 2.3 μm, The resulting histograms showed anticipated distributions. Microfluidic based devices are commonly used for probing single cells, however, very few can determine the cell size. Our technique combines ultrasound and microfluidics to accurately determine the cell size on a cell-by-cell basis, with the potential for integration with fluorescence, light scattering and quantitative photoacoustic techniques for a multiparameter cellular characterization method.

In this study, a phase-change contrast agent for photoacoustic (PA) imaging has been developed and tested invivo using a mouse xenograft tumor model. This agent consists of gold nanorods (GNRs) and a perfluorohexane liquid core and a biodegradable polymer PLGA shell. The agent was conjugated to an anti-HER2 antibody (Herceptin) for specific binding to breast cancer cells which overexpress HER2 receptors. The mean size of the nanoparticles (NPs) was about 285 nm. The targeting specificity of PLGA-GNRs was examined using HER2-positive human breast cancer cells (BT474). The targeted and non-targeted PLGA-GNRs were injected intravenously into Balb/c mice bearing subcutaneous BT474 tumors. Tumors were imaged at different time points using the 21 MHz linear array transducer from the VevoLAZR2100 system (Fujifilm VisualSonics, Canada). The PA radiofrequency spectra were studied as a function of time post-injection. Metrics such as integrated spectral power were used to compare the impact of tumor targeting. Our results show that there is a significant (15 dB) increase in contrast attributed to the NP targeting with HER2 receptors, and little enhancement (~l.lx) of non-targeted PLGA-GNRs at 6-hour post-injection.

Local drug delivery systems, such as in situ forming implants (ISFIs), allow for sustained elevated drug concentration directly at the tumor site. However, these systems have been challenging to translate into clinical practice due to poor drug penetration through the implant/tumor boundary. Ultrasound (US) has emerged as a popular approach to enhance drug release and cellular drug uptake from nanoparticles, but little work has been done with its use in combination with ISFIs. In this study, ISFIs were intratumorally injected and treated with therapeutic ultrasound (TUS). A significant (p

This paper presents a high-frame rate, 3D synthetic transmit aperture system combined with a micro-beamforming technique to reduce the number of measurement channels $M$ (the product of number of transmissions, $n_{T}$ , and the number of receiving channels in each transmission, $n_{R}$ ). Adjacent elements were combined along both the lateral and the elevational direction into groups by applying proper delays in transmission and receiving to focus the beams at a specific region of interest. After the $M$ channels of RF data were acquired, the frequency-domain spatial response function was used to reconstruct the ultrasound images. We have demonstrated that using this approach with a 9-fold reduction in transmission events and 49-fold reduction in receiving channels can achieve the comparable image quality with standard synthetic transmit aperture using a full array. The approach may help in realizing clinical applications of real-time 3D ultrasound imaging technology using large arrays

We have developed a flow cytometer based on simultaneous detection of ultrasound and photoacoustic waves from individual particles/cells flowing in a microfluidic channel. Our polydimethylsiloxane (PDMS) based hydrodynamic 3-dimensional (3D) flow-focusing microfluidic device contains a cross-junction channel, a micro-needle (ID 100 μm and OD 200 μm) insert, and a 3D printed frame to hold and align a high frequency (center frequency 375 MHz) ultrasound transducer. The focused flow passes through a narrow focal zone with lateral and axial focal lengths of 6-8 μm and 15-20 μm, respectively. Both the lateral and axial alignments are achieved by screwing the transducer to the frame onto the PDMS device. Individual particles pass through an interrogation zone in the microfluidic channel with a collinearly aligned ultrasound transducer and a focused 532 nm wavelength laser beam. The particles are simultaneously insonified by high-frequency ultrasound and irradiated by a laser beam. The ultrasound backscatter and laser generated photoacoustic waves are detected for each passing particle. The backscattered ultrasound and photoacoustic signal are strongly dependent on the size, morphology, mechanical properties, and material properties of the flowing particles; these parameters can be extracted by analyzing unique features in the power spectrum of the signals. Frequencies less than 100 MHz do not have these unique spectral signatures. We show that we can reliably distinguish between different particles in a sample using the acoustic-based flow cytometer. This technique, when extended to biomedical applications, allows us to rapidly analyze the spectral signatures from individual single cells of a large cell population, with applications towards label-free detection and characterization of healthy and diseased cells.

Zebrafish are an attractive animal model for the study of disease due to their low cost, ease of care, and ability to rapidly produce large colonies. Some mutant zebrafish, for example those from the casper line, exhibit optical properties which make them ideal specimens for in vivo optical imaging. In this study we use an ultra-high frequency photoacoustic (PA) microscope to image the head and trunk of live casper fish, and generate 3D renderings of the local functional vasculature with single-cell resolution. Five day-post-fertilization larvae were anesthetized using tricane and embedded in 1.5% (w/v) agarose to prevent movement during image acquisition. A transmission mode PA microscope equipped with a single element transducer (either 200 or 400 MHz central frequency) and a 532 nm laser focused through a 4X optical objective was used to raster scan the larvae. Reconstructions of the vasculature were created from the recorded RF data. Images of the trunk showed the caudal artery (CA) and vein (CV), as well as intersegmental vessels (ISV). The ISVs were observed to originate from either the CA or CV, and curve around the notochord to join at the dorsal longitudinal anastomotic vessel. Furthermore, individual erythrocytes were resolved within the caudal hematopoietic tissue. Reconstructions of the head revealed a tortuous organization of developing vessels in the brain. This work is the first demonstration of in vivo PAM at frequencies greater than 200 MHz, and paves the way for future studies which will explore label-free imaging of nanoparticles and cancer progression in vivo.

Photoacoustic (PA) signals carry information of the absorbing chromophores and the light distribution in imaged samples. The dependence of light distribution with optical wavelength affects the accuracy in PA chromophore quantification. Oxygen saturation (sO2) estimations maybe inaccurate in-depth due to the lack of proper fluence compensation. We propose the use of the PA radiofrequency spectral slope (SS) to generate a frequency filter to match the fluence across optical wavelengths. The SS is calculated from the ratio of the radiofrequency power spectra at the selected optical wavelengths. The SS relays information about the absorbers’ size and the light distribution. At the imaged optical wavelengths of the same sample, the SS-estimated size should in principle remain unchanged. This suggests that any changes in the measured SS as a function of optical wavelength can be attributed to the light distribution. A frequency filter can be designed from the computed SS and applied to compensate the PA images. A 5mm phantom consisting of fresh blood, intralipid and gelatin was imaged using the VevoLAZR system at 750 and 850nm. A square sliding window sized 1.6mm with 80% overlap is applied to segment the generated radiofrequency signals. The designed ultrasound filter was applied to each segmented signal. As a result, the fluence-induced depth fluctuations in the sO2 estimations dropped from 9.49%/mm to 1.83%/mm. This will allow for more accurate sO2 estimates that are less depth dependent. The approach provides a new perspective for fluence compensation which can aid in improving chromophore quantification using PA imaging.

Decellularization is a technique that permits the removal of cells from intact tissue while preserving the extracellular matrix structure. It has many applications in the fields of regenerative medicine and tissue engineering. Evaluating the efficiency of cell removal from the tissue non-invasively remains a challenge. This work aims to investigate the use of the k-nearest neighbor classifier using envelope statistics, for the differentiation between freshly excised and decellularized rat kidneys. Two probability distributions were fitted with the histogram of the ultrasound backscatter signal envelope intensities: the Rayleigh and Generalized Gamma. Three fit parameters were extractedfrom these distributions andfed into the classifier. The classification resulted in an AUC of 0.93 and an accuracy of 92%. Future work include incorporating other distributions to further improve the accuracy of this classifier, as well as investigating other classifiers of interest.

Ischemic reperfusion injuries (IRIs) are caused by return of blood to a tissue or organ after a period without oxygen or nutrients. Damage in the microvasculature causes an inflammatory response and heterogeneous scarring, which is associated with an increase in collagen in the extracellular matrix. Although most often associated with heart attacks and strokes, IRI also occurs when blood reperfuses a transplanted organ. Currently, monitoring for IRI is limited to biopsies, which are invasive and sample a limited area. In this work, we explored photoacoustic (PA) biomarkers of scarring. IRI events were induced in mice (n=2) by clamping the left renal artery, then re-establishing flow. At 53 days post-surgery, kidneys were saline perfused and cut in half laterally. One half was immediately imaged with a VevoX system (Fujifilm-VisualSonics, Toronto) in two near infrared ranges - 680 to 970 nm (NIR), and 1200 to 1350 nm (NIR II). The other half was decellularized and then imaged at NIR and NIR II. Regions of interest were manually identified and analyzed for each kidney. For both cellularized and decellularized samples, the PA signal ratio based on irradiation wavelengths of 715:930 nm was higher in damaged kidneys than for undamaged kidneys (p < 0.0001 for both). Damaged kidneys had ROIs with spectra indicating the presence of collagen in the NIR II range, while healthy kidneys did not. Collagen rich spectra were more apparent in decellularized kidneys, suggesting that in the cellularized samples, other components may be contributing to the signal. PA imaging using spectral ratios associated with collagen signatures may provide a non-invasive tool to determine areas of tissue damage due to IRIs.

Ischemic reperfusion injuries (IRIs) occur after blood returns to a tissue or organ after a period without oxygen or nutrients, which causes an inflammatory response leading to heterogeneous scarring of the nearby tissue and vasculature. This is associated with long-term decreases blood flow, and necrosis. Although most commonly associated with heart attacks and strokes, IRIs are also a side effect of organ transplants, when the organ is reperfused in the recipient’s body after being transported from the donor to the transplant hospital. Currently, the optimal method of monitoring for IRI is limited to biopsies, which are invasive and poorly monitor the spatial heterogeneity of the damage. To non-invasively identify changes in kidneys, the left renal artery in mice (n=3) was clamped for 45 minutes to create an IRI event. Both kidneys of each animal were monitored using photoacoustics (PA) with the VevoLAZR system (Fujifilm-VisualSonics, Toronto) three, four and eight weeks after surgery. IRI-treated kidneys show increased picosirius red staining, indicative of collagen (0.601 vs 0.042, p < 0.0001), decreased size as assessed by cross-sectional area (7.8 mm2 vs 35.9 mm2 , p < 0.0001), and decreased oxygen saturation (sO2; 62% vs 77%, p = 0.02). Analysis of the photoacoustic data shows that a two-point metric, the 715:930 nm ratio of the whole kidney (1.05 vs 0.57, p = 0.049) and the optical spectral slope (OSS) (0.8 * 10-3 vs 3.0 * 10-3, p = 0.013) are both able to differentiate between IRI-treated and healthy kidneys. These data suggest that photoacoustics can be used as a non-invasive method to observe in vivo changes in the kidney due to IRI.

Blood transfusion is a life-saving clinical procedure. With millions of units needed globally each year, it is a growing concern to improve product quality and recipient outcomes.Stored red blood cells (RBCs) undergo continuous degradation, leading to structural and biochemical changes. To analyze RBC storage lesions, complex biochemical and biophysical assays are often employed.We demonstrate that label-free imaging flow cytometry and deep learning can characterize RBC morphologies during 42-day storage, replacing the current practice of manually quantifying a blood smear from stored blood units. Based only on bright field and dark field images, our model achieved 90% accuracy in classifying six different RBC morphologies associated with storage lesions versus human-curated manual examination. A model fitted to the deep learning-extracted features revealed a pattern of morphological changes within the aging blood unit that allowed predicting the expiration date of stored blood using solely morphological assessment.Deep learning and label-free imaging flow cytometry could therefore be applied to reduce complex laboratory procedures and facilitate robust and objective characterization of blood samples.

Advances in ultrasound technology have fueled the emergence of Point-Of-Care Ultrasound (PoCU) imaging, including improved ease-of-use, superior image quality, and lower cost ultrasound. One of the approaches that can make the adoption of PoCU universal is to make the data acquisition module as simple as a "stethoscope" while further processing and image construction can be done using cloud-based processors. Toward this goal, we use Structurally Random Matrices (SRM) for compressive sensing of ultrasound data, Fourier sparsifying matrix for recovery in 1D, and frequency domain approach for 2D ultrasound image reconstruction. This approach is demonstrated through wire phantom and in vivo carotid arteries data from ultrasound system using 25%, 12.5%, and 6.25% of the full data rate and ultrasound images of similar perceived quality quantified by Structural Similarity Index Metric (SSIM).