Imaging of renal fibrosis.

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.

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

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

Use of photoacoustic imaging for monitoring vascular disrupting cancer treatments.

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.

Evolving Medical Imaging Techniques for the Assessment of Delayed Graft Function: A Narrative Review.

PURPOSE OF REVIEW: 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. SOURCES OF INFORMATION: PubMed, Google Scholar, and ClinicalTrial.gov up until February 2021. METHODS: 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). KEY FINDINGS: 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. LIMITATIONS: 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.

MOTIF DE LA REVUE: 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. SOURCES: PubMed, Google Scholar et ClinicalTrial.gov jusqu'à février 2021. MÉTHODOLOGIE: 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. PRINCIPAUX RÉSULTATS: 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. LIMITES: 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 vivo photoacoustic assessment of the oxygen saturation changes in the human radial artery: a preliminary study associated with age.

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.

In vivo spectroscopic photoacoustic imaging and laser-induced nanoparticle vaporization for anti-HER2 breast cancer.

This study reports on the development and application of theragnostic agents targeting the HER2 receptors in breast tumors. The agent was constructed by loading silica-coated gold nanorods (GNRs) and a perfluorohexane liquid into PLGA-PEG nanoparticles, followed by surface conjugation with antibody Herceptin. The particle uptake in human breast cancer MDA-MB-231 (HER2-negative) and BT474 (HER2-positive) cell lines was tested. A proof of principle in vivo study was also performed using a xenograft mouse bilateral tumor model (16 mice, 32 tumors). Photoacoustic imaging was performed using a VevoLAZR device at 720/750/850 nm illuminations and 21 MHz central frequency. The relative concentrations of GNRs in the tumor were quantified using a linear spectral unmixing technique. The therapeutic efficacy of these nanoparticles was evaluated through optical droplet vaporization, and cell damage was confirmed using tissue immunofluorescence and histology. Our results demonstrate the potential of PLGA-GNRs as theragnostic agents for anti-HER2 breast cancer therapy.

Fluence-matching technique using photoacoustic radiofrequency spectra for improving estimates of oxygen saturation.

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 imaging biomarkers for monitoring biophysical changes during nanobubble-mediated radiation treatment.

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.

Mean Scatterer Spacing Estimation Using Cepstrum-Based Continuous Wavelet Transform.

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- [Formula: see text] 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 [Formula: see text]) 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.

Photoacoustic imaging of kidney fibrosis for assessing pretransplant organ quality.

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.

Photoacoustic simulations of microvascular bleeding: spectral analysis and its application for monitoring vascular-targeted treatments.

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

Optical and photoacoustic radiofrequency spectroscopic analysis for detecting red blood cell death.

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.

Quantitative Ultrasound Imaging for the Differentiation between Fresh and Decellularized Mouse Kidneys.

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.

Insights into photoacoustic speckle and applications in tumor characterization.

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.

In vitro photoacoustic spectroscopy of pulsatile blood flow: Probing the interrelationship between red blood cell aggregation and oxygen saturation.

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.

Photoacoustic signal characterization of cancer treatment response: Correlation with changes in tumor oxygenation.

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 (<20 s) when the tumor was exposed to mild hyperthermia (43 °C). 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.

Correction: Photoacoustic Imaging of Cancer Treatment Response: Early Detection of Therapeutic Effect from Thermosensitive Liposomes.

[This corrects the article DOI: 10.1371/journal.pone.0165345.].

Simultaneous assessment of red blood cell aggregation and oxygen saturation under pulsatile flow using high-frequency photoacoustics.

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.

Photoacoustic Imaging of Cancer Treatment Response: Early Detection of Therapeutic Effect from Thermosensitive Liposomes.

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

Photoacoustic ultrasound spectroscopy for assessing red blood cell aggregation and oxygenation.

Red blood cell (RBC) aggregation and oxygenation are important markers for a variety of blood disorders. No current technique is capable of simultaneously measuring aggregation/oxygenation levels noninvasively. We propose using photoacoustic ultrasound spectroscopy (PAUS) for assessing both phenomena. This technique relies on frequency-domain analysis of the PA signals by extracting parameters such as the ultrasound spectral slope and the midband fit. To investigate the effect of hematocrit, aggregation, and oxygenation levels on PAUS parameters, a Monte Carlo-based theoretical model and an experimental protocol using porcine RBCs were developed. The samples were illuminated at 750 and 1064 nm and changes in the PAUS parameters were compared to the oxygen-dependent optical absorption coefficients to assess the oxygenation level. Good agreement between the theoretical and experimental spectral parameters was obtained for the spectral slope of the nonaggregated spectra (≈ 0.3 dB/MHz). The experimental midband fit increased by ≈ 5 dB for the largest aggregate size. Based on the analysis of the PA signals, the oxygen saturation level of the most aggregated sample was >20% greater than the nonaggregated sample. The results provide a framework for using PA signals' spectroscopic parameters for monitoring the aggregation and oxygenation levels of RBCs.