Photoacoustics resolves species-specific differences in hemoglobin concentration and oxygenation.

SIGNIFICANCE: Photoacoustic imaging (PAI) enables the detection of blood hemoglobin (HB) concentration and oxygenation (sO2) with high contrast and resolution. Despite the heavy use of photoacoustically determined total hemoglobin (THb) and oxygenation (sO2) biomarkers in PAI research, their relationship with underlying biochemical blood parameters and the impact of intra- and interspecies genetic variability have yet to be established. AIM: To explore the relationship between THb and sO2 photoacoustic biomarkers and the underlying biochemical blood parameters in a species-specific manner. APPROACH: Experiments were performed on blood in vitro using tissue-mimicking agar phantoms. Blood was extracted from mouse, rat, human, and naked mole-rat (Heterocephalus glaber), anticoagulated in ethylenediaminetetraacetic acid, and measured within 48 h. THb and sO2 were measured using a commercial photoacoustic tomography system (InVision 128, iThera Medical GmBH). Biochemical blood parameters such as HB concentration (g/dL), hematocrit (HCT, %), and red blood cell (RBC) count (µL - 1) were assessed using a hematology analyzer (Mythic 18 Vet, Woodley Equipment). RESULTS: A significant correlation was observed between THb and biochemical HB, HCT, and RBC in mouse and rat blood. Moreover, PAI accurately recapitulated interspecies variations in HB and HCT between mouse and rat blood and resolved differences in the oxygen dissociation curves measured using sO2 between human, mouse, and rat. With these validation data in hand, we applied PAI to studies of blood obtained from naked mole-rats and could confirm the high oxygen affinity of this species in comparison to other rodents of similar size. CONCLUSIONS: Our results demonstrate the high sensitivity of photoacoustically determined hemoglobin biomarkers toward species-specific variations in vitro.

Development of a blood oxygenation phantom for photoacoustic tomography combined with online pO2 detection and flow spectrometry (Erratum).

The erratum corrects an error in the article "Development of a blood oxygenation phantom for photoacoustic tomography combined with online pO2 detection and flow spectrometry."

Development of a blood oxygenation phantom for photoacoustic tomography combined with online pO2 detection and flow spectrometry.

Photoacoustic tomography (PAT) is intrinsically sensitive to blood oxygen saturation (sO2) in vivo. However, making accurate sO2 measurements without knowledge of tissue- and instrumentation-related correction factors is extremely challenging. We have developed a low-cost flow phantom to facilitate validation of PAT systems. The phantom is composed of a flow circuit of tubing partially embedded within a tissue-mimicking material, with independent sensors providing online monitoring of the optical absorption spectrum and partial pressure of oxygen in the tube. We first test the flow phantom using two small molecule dyes that are frequently used for photoacoustic imaging: methylene blue and indocyanine green. We then demonstrate the potential of the phantom for evaluating sO2 using chemical oxygenation and deoxygenation of blood in the circuit. Using this dynamic assessment of the photoacoustic sO2 measurement in phantoms in relation to a ground truth, we explore the influence of multispectral processing and spectral coloring on accurate assessment of sO2. Future studies could exploit this low-cost dynamic flow phantom to validate fluence correction algorithms and explore additional blood parameters such as pH and also absorptive and other properties of different fluids.

Photoacoustic imaging as a tool to probe the tumour microenvironment.

The tumour microenvironment (TME) is a complex cellular ecosystem subjected to chemical and physical signals that play a role in shaping tumour heterogeneity, invasion and metastasis. Studying the roles of the TME in cancer progression would strongly benefit from non-invasive visualisation of the tumour as a whole organ in vivo, both preclinically in mouse models of the disease, as well as in patient tumours. Although imaging techniques exist that can probe different facets of the TME, they face several limitations, including limited spatial resolution, extended scan times and poor specificity from confounding signals. Photoacoustic imaging (PAI) is an emerging modality, currently in clinical trials, that has the potential to overcome these limitations. Here, we review the biological properties of the TME and potential of existing imaging methods that have been developed to analyse these properties non-invasively. We then introduce PAI and explore the preclinical and clinical evidence that support its use in probing multiple features of the TME simultaneously, including blood vessel architecture, blood oxygenation, acidity, extracellular matrix deposition, lipid concentration and immune cell infiltration. Finally, we highlight the future prospects and outstanding challenges in the application of PAI as a tool in cancer research and as part of a clinical oncologist's arsenal.

Processing methods for photoacoustic Doppler flowmetry with a clinical ultrasound scanner.

Photoacoustic flowmetry (PAF) based on time-domain cross correlation of photoacoustic signals is a promising technique for deep tissue measurement of blood flow velocity. Signal processing has previously been developed for single element transducers. Here, the processing methods for acoustic resolution PAF using a clinical ultrasound transducer array are developed and validated using a 64-element transducer array with a -6 dB detection band of 11 to 17 MHz. Measurements were performed on a flow phantom consisting of a tube (580 µm inner diameter) perfused with human blood flowing at physiological speeds ranging from 3 to 25 mm / s. The processing pipeline comprised: image reconstruction, filtering, displacement detection, and masking. High-pass filtering and background subtraction were found to be key preprocessing steps to enable accurate flow velocity estimates, which were calculated using a cross-correlation based method. In addition, the regions of interest in the calculated velocity maps were defined using a masking approach based on the amplitude of the cross-correlation functions. These developments enabled blood flow measurements using a transducer array, bringing PAF one step closer to clinical applicability.

Optoacoustics delineates murine breast cancer models displaying angiogenesis and vascular mimicry.

BACKGROUND: Optoacoustic tomography (OT) of breast tumour oxygenation is a promising new technique, currently in clinical trials, which may help to determine disease stage and therapeutic response. However, the ability of OT to distinguish breast tumours displaying different vascular characteristics has yet to be established. The aim of the study is to prove OT as a sensitive technique for differentiating breast tumour models with manifestly different vasculatures. METHODS: Multispectral OT (MSOT) was performed in oestrogen-dependent (MCF-7) and oestrogen-independent (MDA-MB-231) orthotopic breast cancer xenografts. Total haemoglobin (THb) and oxygen saturation (SO2MSOT) were calculated. Pathological and biochemical evaluation of the tumour vascular phenotype was performed for validation. RESULTS: MCF-7 tumours show SO2MSOT similar to healthy tissue in both rim and core, despite significantly lower THb in the core. MDA-MB-231 tumours show markedly lower SO2MSOT with a significant rim-core disparity. Ex vivo analysis revealed that MCF-7 tumours contain fewer blood vessels (CD31+) that are more mature (CD31+/aSMA+) than MDA-MB-231. MCF-7 presented higher levels of stromal VEGF and iNOS, with increased NO serum levels. The vasculogenic process observed in MCF-7 was consistent with angiogenesis, while MDA-MB-231 appeared to rely more on vascular mimicry. CONCLUSIONS: OT is sensitive to differences in the vascular phenotypes of our breast cancer models.

Photoacoustic imaging using genetically encoded reporters: a review.

Genetically encoded contrast in photoacoustic imaging (PAI) is complementary to the intrinsic contrast provided by endogenous absorbing chromophores such as hemoglobin. The use of reporter genes expressing absorbing proteins opens the possibility of visualizing dynamic cellular and molecular processes. This is an enticing prospect but brings with it challenges and limitations associated with generating and detecting different types of reporters. The purpose of this review is to compare existing PAI reporters and signal detection strategies, thereby offering a practical guide, particularly for the nonbiologist, to choosing the most appropriate reporter for maximum sensitivity in the biological and technological system of interest.

Evaluation of Precision in Optoacoustic Tomography for Preclinical Imaging in Living Subjects.

Optoacoustic tomography (OT) is now widely used in preclinical imaging; however, the precision (repeatability and reproducibility) of OT has yet to be determined. Methods: We used a commercial small-animal OT system. Measurements in stable phantoms were used to independently assess the impact of system variables on precision (using coefficient of variation, COV), including acquisition wavelength, rotational position, and frame averaging. Variables due to animal handling and physiology, such as anatomic placement and anesthesia conditions, were then assessed in healthy nude mice using the left kidney and spleen as reference organs. Temporal variation was assessed by repeated measurements over hours and days both in phantoms and in vivo. Sensitivity to small-molecule dyes was determined in phantoms and in vivo; precision was assessed in vivo using IRDye800CW. Results: OT COV in a stable phantom was less than 2.8% across all wavelengths over 30 d. The factors with the greatest impact on signal repeatability in phantoms were rotational position and user experience, both of which still resulted in a COV of less than 4% at 700 nm. Anatomic region-of-interest size showed the highest variation, at 12% and 18% COV in the kidney and spleen, respectively; however, functional SO2 measurements based on a standard operating procedure showed an exceptional reproducibility of less than 4% COV. COV for repeated injections of IRDye800CW was 6.6%. Sources of variability for in vivo data included respiration rate, degree of user experience, and animal placement. Conclusion: Data acquired with our small-animal OT system were highly repeatable and reproducible across subjects and over time. Therefore, longitudinal OT studies may be performed with high confidence when our standard operating procedure is followed.

Towards Quantitative Evaluation of Tissue Absorption Coefficients Using Light Fluence Correction in Optoacoustic Tomography.

Optoacoustic tomography is a fast developing imaging modality, combining the high contrast available from optical excitation of tissue with the high resolution and penetration depth of ultrasound detection. Light is subject to both absorption and scattering when traveling through tissue; adequate knowledge of tissue optical properties and hence the spatial fluence distribution is required to create an optoacoustic image that is directly proportional to chromophore concentrations at all depths. Using data from a commercial multispectral optoacoustic tomography (MSOT) system, we implemented an iterative optimization for fluence correction based on a finite-element implementation of the delta-Eddington approximation to the Radiative Transfer Equation (RTE). We demonstrate a linear relationship between the image intensity and absorption coefficients across multiple wavelengths and depths in phantoms. We also demonstrate improved feature visibility and spectral recovery at depth in phantoms and with in vivo measurements, suggesting our approach could in the future enable quantitative extraction of tissue absorption coefficients in biological tissue.

Velocity measurements in whole blood using acoustic resolution photoacoustic Doppler.

Acoustic resolution photoacoustic Doppler velocimetry promises to overcome the spatial resolution and depth penetration limitations of current blood flow measuring methods. Despite successful implementation using blood-mimicking fluids, measurements in blood have proved challenging, thus preventing in vivo application. A common explanation for this difficulty is that whole blood is insufficiently heterogeneous relative to detector frequencies of tens of MHz compatible with deep tissue photoacoustic measurements. Through rigorous experimental measurements we provide new insight that refutes this assertion. We show for the first time that, by careful choice of the detector frequency and field-of-view, and by employing novel signal processing methods, it is possible to make velocity measurements in whole blood using transducers with frequencies in the tens of MHz range. These findings have important implications for the prospects of making deep tissue measurements of blood flow relevant to the study of microcirculatory abnormalities associated with cancer, diabetes, atherosclerosis and other conditions.

Acoustic resolution photoacoustic Doppler velocimetry in blood-mimicking fluids.

Photoacoustic Doppler velocimetry provides a major opportunity to overcome limitations of existing blood flow measuring methods. By enabling measurements with high spatial resolution several millimetres deep in tissue, it could probe microvascular blood flow abnormalities characteristic of many different diseases. Although previous work has demonstrated feasibility in solid phantoms, measurements in blood have proved significantly more challenging. This difficulty is commonly attributed to the requirement that the absorber spatial distribution is heterogeneous relative to the minimum detectable acoustic wavelength. By undertaking a rigorous study using blood-mimicking fluid suspensions of 3 µm absorbing microspheres, it was discovered that the perceived heterogeneity is not only limited by the intrinsic detector bandwidth; in addition, bandlimiting due to spatial averaging within the detector field-of-view also reduces perceived heterogeneity and compromises velocity measurement accuracy. These detrimental effects were found to be mitigated by high-pass filtering to select photoacoustic signal components associated with high heterogeneity. Measurement under-reading due to limited light penetration into the flow vessel was also observed. Accurate average velocity measurements were recovered using "range-gating", which furthermore maps the cross-sectional velocity profile. These insights may help pave the way to deep-tissue non-invasive mapping of microvascular blood flow using photoacoustic methods.

Pulsed photoacoustic Doppler flowmetry using time-domain cross-correlation: accuracy, resolution and scalability.

The feasibility of making spatially resolved measurements of blood velocity using a pulsed photoacoustic Doppler technique in acoustic resolution mode has been investigated. Doppler time shifts were quantified via cross-correlation of photoacoustic waveform pairs generated within a blood-simulating phantom using pairs of light pulses. The phantom comprised micron-scale absorbers imprinted on an acetate sheet and moved at known velocities. The photoacoustic waves were detected using PZT ultrasound transducers operating at center frequencies of 20 MHz, 5 MHz and 3.5 MHz; measurements of velocity and resolution were calculated from the mean cross-correlation function of 25 waveform pairs. Velocities in the range ±0.15 to ±1.5 ms(-1) were quantified with accuracies as low as 1%. The transducer focal beam width determines a maximum measurable velocity |V(max)| beyond which correlation is lost due to absorbers moving out of the focal beam between the two laser pulses. Below |V(max)| a measurement resolution of <4% of the measured velocity was achieved. Resolution and |V(max)| can be scaled to much lower velocities such as those encountered in microvasculature (< 50 mms(-1)). The advantage of pulsed rather than continuous-wave excitation is that spatially resolved velocity measurements can be made, offering the prospect of mapping flow within the microcirculation.

An electrically-activated dynamic tissue-equivalent phantom for assessment of diffuse optical imaging systems.

A novel design of solid dynamic phantom with tissue-like optical properties is presented, which contains variable regions of contrast which are activated electrically. Reversible changes in absorption are produced by localized heating of targets impregnated with thermochromic pigment. A portable, battery-operated prototype has been constructed, and its optical and temporal characteristics have been investigated. The phantom has been developed as a means of assessing the performance of diffuse optical imaging systems, such as those used to monitor haemodynamic changes in the brain and other tissues. Images of the phantom have been reconstructed using data acquired with a continuous wave optical topography system.