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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 singleelement transducers with central frequencies ranging from 40–400MHz. 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.

Frequency analysis of the photoacoustic radiofrequency signals and oxygen saturation estimates were used to monitor the in-vivo response of a novel, thermosensitive liposome treatment. The liposome encapsulated doxorubicin (HaT-DOX) releasing it rapidly (Photoacoustic imaging (VevoLAZR, 750/850 nm, 40 MHz) of EMT-6 breast cancer tumors was performed 30 min pre- and post-treatment and up to 7 days post-treatment (at 2/5/24 h timepoints). HaT-DOX-treatment responders exhibited on average a 22% drop in oxygen saturation 2 h post-treatment and a decrease (45% at 750 nm and 73% at 850 nm) in the slope of the normalized PA frequency spectra. The spectral slope parameter correlated with treatment-induced hemorrhaging which increased the optical absorber effective size via interstitial red blood cell leakage. Combining frequency analysis and oxygen saturation estimates differentiated treatment responders from non-responders/control animals by probing the treatment-induced structural changes of blood vessel.

We investigate the feasibility of photoacoustic (PA) imaging for assessing the correlation between red blood cell (RBC) aggregation and the oxygen saturation (sO2) in a simulated pulsatile blood flow system. For the 750 and 850 nm illuminations, the PA amplitude (PAA) increased and decreased as the mean blood flow velocity decreased and increased, respectively, at all beat rates (60, 120 and 180 bpm). The sO2 also cyclically varied, in phase with the PAA for all beat rates. However, the linear correlation between the sO2 and the PAA at 850 nm was stronger than that at 750 nm. These results suggest that the sO2 can be correlated with RBC aggregation induced by decreased mean shear rate in pulsatile flow, and that the correlation is dependent on the optical wavelength. The hemodynamic properties of blood flow assessed by PA imaging may be used to provide a new biomarker for simultaneous monitoring blood viscosity related to RBC aggregation, oxygen delivery related to the sO2 and their clinical correlation.

Roughly 10% of the world’s population has chronic kidney disease (CKD). In its advanced stages, CKD greatly increases the risk of hospitalization and death. Although kidney transplantation has revolutionized the care of advanced CKD, clinicians have limited ways of assessing donor kidney quality. Thus, optimal donor kidney–recipient matching cannot be performed, meaning that some patients receive damaged kidneys that function poorly. Fibrosis is a form of chronic damage often present in donor kidneys, and it is an important predictor of future renal function. Currently, no safe, easy-to-perform technique exists that accurately quantifies renal fibrosis. We describe a potentially novel photoacoustic (PA) imaging technique that directly images collagen, the principal component of fibrotic tissue. PA imaging noninvasively quantifies whole kidney fibrotic burden in mice, and cortical fibrosis in pig and human kidneys, with outstanding accuracy and speed. Remarkably, 3-dimensional PA imaging exhibited sufficiently high resolution to capture intrarenal variations in collagen content. We further show that PA imaging can be performed in a setting that mimics human kidney transplantation, suggesting the potential for rapid clinical translation. Taken together, our data suggest that PA collagen imaging is a major advance in fibrosis quantification that could have widespread preclinical and clinical impact.

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

This dissertation describes the development of functional and structural photoacoustic (PA) imaging biomarkers that can be used to monitor cancer treatment response and potentially predict treatment outcome. An imaging method that can indicate individualized treatment success could improve therapeutic outcome. Assessing the effectiveness of therapies as early as possible may spare the patient from unnecessary treatments and save precious clinical resources. In order for PA imaging to enter mainstream radiology and become a treatment monitoring tool, rigorous development of biomarkers that are easy-to-use and representative of the treatment-induced changes in the tumor microenvironment are needed. In this work, I have developed imaging biomarkers that rely on the analysis of the radiofrequency signals in acoustic resolution PA imaging. Specifically, I show through simulations and experiments that biomarkers sensitive to the size, number density and spacing of tumor blood vessels can be extracted through time and frequency domain analysis of PA signals. This information is encoded in the speckle that forms during diffuse optical illumination, which was previously thought to be noise. Moreover, I demonstrate that PA imaging can detect the response of a thermosensitive liposome by measuring a >10% drop in the oxygenation of the tumor as early as 30 minutes post-treatment. This change in oxygenation is due to vascular disruption, a phenomenon that can be detected through frequency analysis of the PA signals. The spectral slope parameter decreases by as much as 73% in 2 hours post-treatment and can be used to differentiate alongside the oxygenation biomarker between responders and non-responders. Lastly, I demonstrate that these PA imaging biomarkers correlate well with the histologically measured biophysical changes of two novel, bubble-based cancer treatments. In this dissertation, PA imaging biomarkers for cancer treatment monitoring are developed, advancing the modality towards clinical translation.

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

The potential of photoacoustic imaging for detecting red blood cell (RBC) aggregation is explored. Enhanced aggregation is observed in disorders such as diabetes impairing oxygen release into tissue. Simultaneous measurements of aggregation and oxygenation levels cannot be made using current tools. Photoacoustic detection of aggregation and assessment of oxygen saturation was investigated. A theoretical and experimental model of aggregation was developed using human and porcine RBCs. Frequency-domain analysis of the PA signals was used to derive the spectral slope and midband fit of the normalized power spectra for various hematorit and aggregation conditions. Oxygen saturation was assessed using multiple wavelengths of illumination. The experimental spectral slope (~0.3 dB/MHz) for non-aggregated samples agreed with the theory decreasing with increasing aggregate size. The midband fit increased by ~5 dB when the aggregate size reached the largest level while the oxygen saturation increased by > 20%. These results suggest that photoacoustic-radio-frequency-spectroscopic-parameters have the potential to monitor RBC aggregation and oxygenation level.

Delayed graft function (DGF) is a significant complication that contributes to poorer graft function and shortened graft survival. In this review, we sought to evaluate the current and emerging role of medical imaging modalities in the assessment of DGF and how it may guide clinical management.PubMed, Google Scholar, and ClinicalTrial.gov up until February 2021.This narrative review first examined the pathophysiology of DGF and current clinical management. We then summarized relevant studies that utilized medical imaging to assess posttransplant renal complications, namely, DGF. We focused our attention on noninvasive, evolving imaging modalities with the greatest potential for clinical translation, including contrast-enhanced ultrasound (CEUS) and multiparametric magnetic resonance imaging (MRI).A kidney biopsy in the setting of DGF can be used to assess the degree of ischemic renal injury and to rule out acute rejection. Biopsies are accompanied by complications and may be limited by sampling bias. Early studies on CEUS and MRI have shown their potential to distinguish between the 2 most common causes of DGF (acute tubular necrosis and acute rejection), but they have generally included only small numbers of patients and have not kept pace with more recent technical advances of these imaging modalities. There remains unharnessed potential with CEUS and MRI, and more robust clinical studies are needed to better evaluate their role in the current era.The adaptation of emerging approaches for imaging DGF will depend on additional clinical trials to study the feasibility and diagnostic test characteristics of a given modality. This is limited by access to devices, technical competence, and the need for interdisciplinary collaborations to ensure that such studies are well designed to appropriately inform clinical decision-making.La reprise retardée de la fonction du greffon (RRFG) est une complication importante susceptible d’affecter négativement la fonction du greffon et de réduire sa survie. Dans cette revue, nous cherchions à évaluer le rôle actuel et grandissant des modalités d’imagerie médicale dans l’évaluation de la RRFG et la façon dont cela pourrait orienter la prise en charge clinique.PubMed, Google Scholar et ClinicalTrial.gov jusqu’à février 2021.Notre revue narrative portait d’abord sur la physiopathologie de la RRFG et la prise en charge clinique actuelle. Nous avons par la suite résumé les études pertinentes ayant utilisé l’imagerie médicale pour évaluer les complications rénales post- transplantation, notamment la RRFG. Nous avons concentré notre attention sur les modalités d’imagerie non effractives et évolutives présentant le plus grand potentiel d’application clinique, notamment l’échographie de contraste (CEUS) et l’imagerie par résonance magnétique (IRM) multiparamétrique.Dans les cas de RRFG, une biopsie du rein peut être utilisée pour évaluer l’ampleur des lésions rénales ischémiques et pour exclure le rejet aigu. Les biopsies s’accompagnent de complications et pourraient être limitées par des biais d’échantillonnage. Des études préliminaires examinant les CEUS et l’IRM ont montré que ces modalités permettaient une distinction entre les deux causes les plus fréquentes de la RRFG (nécrose tubulaire aiguë et rejet aigu), mais ces études portaient généralement sur de petits nombres de patients et n’avaient pas suivi les plus récents progrès techniques de ces modalités d’imagerie. Il subsiste un potentiel non exploité avec les CEUS et l’IRM. Des études cliniques plus robustes sont nécessaires pour mieux évaluer leur rôle à l’heure actuelle.L’adaptation des approches émergentes pour l’imagerie en contexte de RRFG dépendra d’essais cliniques supplémentaires qui examineront la faisabilité et les caractéristiques des tests diagnostiques d’une modalité donnée. Cela est limité par l’accès aux appareils, la compétence technique et la nécessité de collaborations interdisciplinaires afin de s’assurer que ces études sont bien conçues et qu’elles puissent éclairer adéquatement la prise de décisions cliniques.

In the kidney and liver, fibrosis is a powerful predictor of outcomes. Unfortunately, accurate fibrosis quantification cannot be done non-invasively. Photoacoustic (PA) imaging offers the potential for measuring collagen, the principal component of the fibrotic matrix. Here, we demonstrate the potential of PA imaging for fibrosis quantification in preclinical kidney and liver fibrosis models. To induce kidney fibrosis, the left ureter was obstructed for 0 (n = 5), 7 (n = 5) or 14 days (n =5). The right kidneys at each day (n = 15) served as controls. To induce liver fibrosis, carbon tetrachloride (CCl4) injections were given to mice (n = 31). Control mice (n = 12) received corn oil vehicle injections. A PA unmixing algorithm based on the use of the variance inflation coefficient for detecting the multicollinearity of collagen/oxy/deoxyhemoglobin was developed and validated against gold standard histology measurements. The VevoLAZR-X system was used in the 680-930 nm illumination range at 15 MHz. PA imaging revealed 4x more collagen by day 14 post ureteral obstruction compared to sham kidneys. Similarly, PA imaging suggested that fibrotic livers 6 weeks post-CCl4 injection contained about 125% more collagen than control livers. Comparisons with histological gold standards revealed a strong linear correlation (r = 0.89 – 0.99). This work demonstrates the potential of PA imaging to accurately and non-invasively assess fibrotic burden in livers and kidneys. We postulate that this technology will accelerate the preclinical assessment of antifibrotic drugs.

Gas-filled nanobubbles (NBs) have been used for contrast enhanced ultrasound (US) molecular imaging similar to their microbubble (MB) counterparts. In this work we explore the use of NBs as cancer therapeutic vehicles to enhance radiation therapy and examine the role of photoacoustic (PA) imaging for quantifying the tissue response. Experiments were conducted in 58 mice bearing prostate cancer tumors. The treatment groups consisted of untreated controls (n=14), 8Gy radiation (n=14), US+MB (n=8), US+NB (n=5), US+MB+8Gy (n=12) and US+NB+8Gy (n=5). PA imaging was conducted using the VevoLAZR 21 MHz probe at pre-treatment, 2h and 24h post-treatment. To examine the changes in the tumor microenvironment post-treatment, we measured the tumor oxygen saturation (sO2), total hemoglobin (HbT) and the spectral slope (SS) as a surrogate measurement of vessel size. Histology measures of tumor vascularity/vessel size (CD31) and cellular death (TUNEL) allowed us to interpret the PA biomarker changes. The PA biomarkers revealed the following changes for the US+NB+8Gy group: (a) a 20% decrease in tumor sO2, (b) a 30% increase in tHb and (c) 20% decrease SS. These results suggest that NBs induce vessel damage from US-induced cavitation. Histology elucidates these findings: CD31 staining measuring vascularity decreases by 80% at 24h–this causes the drop in sO2 and increase in HbT; the size of vessels decreases by 10%–this is captured by the SS change. This work shows that PA imaging biomarkers can quantify these vascular changes which in turn enhance the radiation-induced cell death (up to 40%, as measured by TUNEL staining).

Significance: We demonstrate the potential of probing the sO2 change under blood flow in vivo using photoacoustic (PA) imaging and sheds light on the complex relationship between RBC aggregation and oxygen delivery. Aim: To conduct in vivo assessments of the sO2 in the radial artery of healthy volunteers and simultaneously probe the relation between the sO2 and hemodynamic behavior such as red blood cell (RBC) aggregation. Approach: The effects of PA-based measurements of blood hemodynamics were studied as a function of the subjects’ age (20s, 30s, and 40s). The pulsatile blood flow in the human radial artery of 12 healthy subjects was imaged in the 700 to 900 nm optical wavelength range using a linear array-based PA system. Results: The PA power when blood velocity is minimum (Pamax) was larger than the one attained at maximum blood velocity (Pamin), consistent with predictions based on the cyclical variation of RBC aggregation during pulsatile flow. The difference between Pamin and Pamax at 800 nm (ΔPa800) increased with age (1.7, 2.2, and 2.6 dB for age group of 20s, 30s, and 40s, respectively). The sO2 computed from Pamax was larger than the one from Pamin. Conclusions: The ΔPa800 increased with participant age. The ΔPa800 metric could be a surrogate of noninvasively monitoring the age-induced changes in RBC aggregation. The sO2 change during a cycle of pulsatile blood flow also increased with age, demonstrating that RBC aggregation can affect the sO2 change.

Graphical abstract, The development of novel anticancer therapies warrants the parallel development of biomarkers that can quantify their effectiveness. Photoacoustic imaging has the potential to measure changes in tumor vasculature during treatment. Establishing the accuracy of imaging biomarkers requires direct comparisons with gold histological standards. In this work, we explore whether a new class of submicron, vascular disrupting, ultrasonically stimulated nanobubbles enhance radiation therapy. In vivo experiments were conducted on mice bearing prostate cancer tumors. Combined nanobubble plus radiation treatments were compared against conventional microbubbles and radiation alone (single 8 Gy fraction). Acoustic resolution photoacoustic imaging was used to monitor the effects of the treatments 2- and 24-hs post-administration. Histological examination provided metrics of tumor vascularity and tumoral cell death, both of which were compared to photoacoustic-derived biomarkers. Photoacoustic metrics of oxygen saturation reveal a 20 % decrease in oxygenation within 24 h post-treatment. The spectral slope metric could separate the response of the nanobubble treatments from the microbubble counterparts. This study shows that histopathological assessment correlated well with photoacoustic biomarkers of treatment response.

PURPOSE OF REVIEW Fibrosis is an important biomarker of chronic kidney injury, and a powerful predictor of renal outcome. Currently, the only method for measuring fibrotic burden is histologic analysis, which requires a kidney biopsy in humans, or kidney removal in animal models. These requirements have not only hindered our ability to manage patients effectively, but have also prevented a full understanding of renal fibrosis pathogenesis, and slowed the translation of new antifibrotic agents. The development of noninvasive fibrosis imaging tools could thus transform both clinical care and renal fibrosis research. RECENT FINDINGS Conventional imaging modalities have historically failed to image fibrosis successfully. However, recent exciting technological advances have greatly enhanced their capabilities. New techniques, for example, may allow imaging of the physical consequences of scarring, as surrogate measures of renal fibrosis. Similarly, other groups have developed ways to directly image extracellular matrix, either with the use of contrast-enhanced probes, or using matrix components as endogenous contrast agents. SUMMARY New developments in imaging technology have the potential to transform our ability to visualize renal fibrosis and to monitor its progression. In doing so, these advances could have major implications for kidney disease care, the development of new antiscarring agents, and our understanding of renal fibrosis in general.

Vascular disrupting agents disrupt tumor vessels, blocking the nutritional and oxygen supply tumors need to thrive. This is achieved by damaging the endothelium lining of blood vessels, resulting in red blood cells (RBCs) entering the tumor parenchyma. RBCs present in the extracellular matrix are exposed to external stressors resulting in biochemical and physiological changes. The detection of these changes can be used to monitor the efficacy of cancer treatments. Spectroscopic photoacoustic (PA) imaging is an ideal candidate for probing RBCs due to their high optical absorption relative to surrounding tissue. The goal of this work is to use PA imaging to monitor the efficacy of the vascular disrupting agent 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) through quantitative analysis. Then, 4T1 breast cancer cells were injected subcutaneously into the left hind leg of eight BALB/c mice. After 10 days, half of the mice were treated with 15 mg/kg of DMXAA and the other half were injected with saline. All mice were imaged using the VevoLAZR X PA system before treatment, 24 and 72 hours after treatment. The imaging was done at six wavelengths and linear spectral unmixing was applied to the PA images to quantify three forms of hemoglobin (oxy, deoxy and met-hemoglobin). After imaging, tumors were histologically processed and H&E and TUNEL staining were used to detect the tissue damage induced by the DMXAA treatment. The total hemoglobin concentration remained unchanged after treatment for the saline treated mice. For DMXAA treated mice, a 10% increase of deoxyhemoglobin concentration was detected 24 hours after treatment and a 22.6% decrease in total hemoglobin concentration was observed by 72 hours. A decrease in the PA spectral slope parameters was measured 24 hours after treatment. This suggests that DMXAA induces vascular damage, causing red blood cells to extravasate. Furthermore, H&E staining of the tumor showed areas of bleeding with erythrocyte deposition. These observations are further supported by the increase in TUNEL staining in DMXAA treated tumors, revealing increased cell death due to vascular disruption. This study demonstrates the capability of PA imaging to monitor tumor vessel disruption by the vascular disrupting agent DMXAA.

In this work, we examine the potential of photoacoustic imaging for understanding the biophysical mechanism of nanobubble and microbubble-based vascular disrupting therapies. We present for the first time, a direct in-vivo comparison between sub-micron nanobubbles and the commercially available microbubbles. Our results show that PA imaging of tumor oxygenation is capable of measuring the nanobubble-induced almost 40% cell death as a result of vascular disruption.

In this work, we present the first time use of photoacoustic imaging for assessing the quality of donor kidneys pre-transplantation. There is a pressing clinical need to quantify the fibrotic (scarring) burden in a non-invasive manner to give clinicians crucial information before they decide whether to accept a donor kidney. Our results in human kidneys show that photoacoustic imaging can be a robust tool for assessing the degree of scarring, an important predictor of post-transplantation clinical outcome.

The goal of this study was to develop an ultrasound (US) scatterer spacing estimation method using an enhanced cepstral analysis based on continuous wavelet transforms (CWTs). Simulations of backscattering media containing periodic and quasi-periodic scatterers were carried out to test the developed algorithm. Experimental data from HT-29 pellets and in vivo PC3 tumors were then used to estimate the mean scatterer spacing. For simulated media containing quasi-periodic scatterers at 1-mm and 100- $\mu \text{m}$ spacing with 5% positional variation, the developed algorithm yielded a spacing estimation error of ~1% for 25- and 55-MHz US pulses. The mean scatterer spacing of HT-29 cell pellets (31.97 $\mu \text{m}$ ) was within 3% of the spacing obtained from histology and agreed with the predicted spacing from simulations based on the same pellets for both frequencies. The agreement extended to in vivo PC3 tumors estimation of the spacing with a variance of 1.68% between the spacing derived from the tumor histology and the application of the CWT to the experimental results. The developed technique outperformed the traditional cepstral methods as it can detect nonprominent peaks from quasi-random scatterer configurations. This work can be potentially used to detect morphological tissue changes during normal development or disease treatment.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.