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The ability of photoacoustic imaging to measure functional tissue properties, such as blood oxygenation sO 2 , enables a wide variety of possible applications. sO 2 can be computed from the ratio of oxyhemoglobin HbO 2 and deoxyhemoglobin Hb, which can be distuinguished by multispectral photoacoustic imaging due to their distinct wavelength-dependent absorption. However, current methods for estimating sO 2 yield inaccurate results in realistic settings, due to the unknown and wavelength-dependent influence of the light fluence on the signal. In this work, we propose learned spectral decoloring to enable blood oxygenation measurements to be inferred from multispectral photoacoustic imaging. The method computes sO 2 pixel-wise, directly from initial pressure spectra S p 0 ( λ , x ) , which represent initial pressure values at a fixed spatial location x over all recorded wavelengths λ . The method is compared to linear unmixing approaches, as well as pO 2 and blood gas analysis reference measurements. Experimental results suggest that the proposed method is able to obtain sO 2 estimates from multispectral photoacoustic measurements in silico, in vitro, and in vivo.

Reactive oxygen species (ROS) are a diverse group of molecules that serve as both essential signalling mediators and potential drivers of oxidative stress. In tumours, ROS influence critical processes such as proliferation, angiogenesis, metabolic adaptation and therapy resistance. These processes are further modulated by reduced oxygen availability (hypoxia), a defining feature of many solid tumours that can alter redox balance and cellular signalling. The interplay between ROS and hypoxia is highly dynamic, with both factors shaping tumour behaviour in complex and often unpredictable ways. Accurately measuring ROS and tumour oxygenation remains a significant challenge due to their transient nature and variability in levels across different tumour types. In this guide, we provide a comprehensive update on the dynamic interaction between ROS and hypoxia in tumours, evaluate current strategies for ROS detection and discuss emerging therapeutic approaches that target redox vulnerabilities in cancer. Understanding the intricate relationship between ROS and hypoxia is crucial for refining therapeutic strategies and improving patient outcomes. Hypoxia reshapes tumour redox landscapes by altering compartmental ROS production (mitochondria, NOX, ER, peroxisomes). Accurate interpretation requires oxygen‐contextualised measurement (live biosensors, chemical probes, EPR, LC–MS) and awareness of artefacts (reoxygenation, probe specificity). Therapeutic strategies either exploit elevated ROS to induce cell death or target antioxidant adaptations, with efficacy critically dependent on oxygenation and temporal dynamics. Created in BioRender. Hammond, E. (2025). https://BioRender.com/w3i8lv9.

BackgroundOptoacoustic 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.MethodsMultispectral 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.ResultsMCF-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.ConclusionsOT is sensitive to differences in the vascular phenotypes of our breast cancer models.

Mesoscopic photoacoustic imaging (PAI) enables label‐free visualization of vascular networks in tissues with high contrast and resolution. Segmenting these networks from 3D PAI data and interpreting their physiological and pathological significance is crucial yet challenging due to the time‐consuming and error‐prone nature of current methods. Deep learning offers a potential solution; however, supervised analysis frameworks typically require human‐annotated ground‐truth labels. To address this, an unsupervised image‐to‐image translation deep learning model is introduced, the Vessel Segmentation Generative Adversarial Network (VAN‐GAN). VAN‐GAN integrates synthetic blood vessel networks that closely resemble real‐life anatomy into its training process and learns to replicate the underlying physics of the PAI system in order to learn how to segment vasculature from 3D photoacoustic images. Applied to a diverse range of in silico, in vitro, and in vivo data, including patient‐derived breast cancer xenograft models and 3D clinical angiograms, VAN‐GAN demonstrates its capability to facilitate accurate and unbiased segmentation of 3D vascular networks. By leveraging synthetic data, VAN‐GAN reduces the reliance on manual labeling, thus lowering the barrier to entry for high‐quality blood vessel segmentation (F1 score: VAN‐GAN vs. U‐Net = 0.84 vs. 0.87) and enhancing preclinical and clinical research into vascular structure and function. This study introduces VAN‐GAN, an unsupervised deep learning model for 3D vascular network segmentation in mesoscopic photoacoustic imaging. By integrating synthetic blood vessel networks and advanced training techniques, VAN‐GAN demonstrates accurate and unbiased segmentation across in silico, in vitro, and in vivo datasets, including clinical angiograms, reducing reliance on manual labeling and minimizing bias in vascular research.

There is a critical unmet clinical need to identify biomarkers that predict and detect radiation therapy (RT) response in cancer. Using the unique capabilities of multi‐scale photoacoustic imaging (PAI) to depict tumor oxygenation and vasculature in vivo, we identified surrogate biomarkers of RT response in two human breast cancer xenograft models (MCF7 and MDA‐MB‐231), comparing hypofractionated delivery with an ablative single‐dose scheme. Mesoscopic and multispectral tomographic PAI were performed 24h pre‐RT, 24h post‐RT and at endpoint and were supported by ex vivo immunohistochemistry. MCF7 xenografts, which exhibited a denser and more mature vasculature, showed improved response to both RT schemes than MDA‐MB‐231, in terms of oxygenation, volume control and proliferation. Higher pre‐RT oxygenation and oxygen‐diffusion capacity were associated with improved outcome, consistent with the oxygen‐enhancement effect. PAI further revealed regimen‐specific vascular effects: ablative RT produced early pruning of looping vessels and superficial blood volume, while only hypofractionated RT led to a rise in intratumoral oxygenation at the endpoint in radiosensitive MCF7, indicative of reduced oxygen consumption in damaged tumor cells. We showed that PAI could capture early RT response and inform on radioresistance, thus demonstrating PAI as a promising tool to monitor the tumor vascular response to RT. Tomographic and mesoscopic photoacoustics capture intratumoural features of radioresistance and response.

A lack of accepted standards and standardized phantoms suitable for the technical validation of biophotonic instrumentation hinders the reliability and reproducibility of its experimental outputs. In this Perspective, we discuss general criteria for the design of tissue-mimicking biophotonic phantoms, and use these criteria and state-of-the-art developments to critically review the literature on phantom materials and on the fabrication of phantoms. By focusing on representative examples of standardization in diffuse optical imaging and spectroscopy, fluorescence-guided surgery and photoacoustic imaging, we identify unmet needs in the development of phantoms and a set of criteria (leveraging characterization, collaboration, communication and commitment) for the standardization of biophotonic instrumentation. This Perspective overviews the design and fabrication of tissue-mimicking phantom materials for the technical validation of biophotonic instrumentation, and identifies a set of criteria for their standardization.

The capillaries are the smallest blood vessels in the body, typically imaged using video capillaroscopy to aid diagnosis of connective tissue diseases, such as systemic sclerosis. Video capillaroscopy allows visualization of morphological changes in the nailfold capillaries but does not provide any physiological information about the blood contained within the capillary network. Extracting parameters such as hemoglobin oxygenation could increase sensitivity for diagnosis and measurement of microvascular disease progression. To design, construct, and test a low-cost multispectral imaging (MSI) system using light-emitting diode (LED) illumination to assess relative hemoglobin oxygenation in the nailfold capillaries. An LED ring light was first designed and modeled. The ring light was fabricated using four commercially available LED colors and a custom-designed printed circuit board. The experimental system was characterized and results compared with the illumination model. A blood phantom with variable oxygenation was used to determine the feasibility of using the illumination-based MSI system for oximetry. Nailfold capillaries were then imaged in a healthy subject. The illumination modeling results were in close agreement with the constructed system. Imaging of the blood phantom demonstrated sensitivity to changing hemoglobin oxygenation, which was in line with the spectral modeling of reflection. The morphological properties of the volunteer capillaries were comparable to those measured in current gold standard systems. LED-based illumination could be used as a low-cost approach to enable MSI of the nailfold capillaries to provide insight into the oxygenation of the blood contained within the capillary network.

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.

Isoprene-epoxydiols-derived secondary organic aerosol (IEPOX-SOA) can contribute substantially to organic aerosol (OA) concentrations in forested areas under low NO conditions, hence significantly influencing the regional and global OA budgets, accounting, for example, for 16–36 % of the submicron OA in the southeastern United States (SE US) summer. Particle evaporation measurements from a thermodenuder show that the volatility of ambient IEPOX-SOA is lower than that of bulk OA and also much lower than that of known monomer IEPOX-SOA tracer species, indicating that IEPOX-SOA likely exists mostly as oligomers in the aerosol phase. The OH aging process of ambient IEPOX-SOA was investigated with an oxidation flow reactor (OFR). New IEPOX-SOA formation in the reactor was negligible, as the OFR does not accelerate processes such as aerosol uptake and reactions that do not scale with OH. Simulation results indicate that adding  ∼  100 µg m−3 of pure H2SO4 to the ambient air allows IEPOX-SOA to be efficiently formed in the reactor. The heterogeneous reaction rate coefficient of ambient IEPOX-SOA with OH radical (kOH) was estimated as 4.0 ± 2.0  ×  10−13 cm3 molec−1 s−1, which is equivalent to more than a 2-week lifetime. A similar kOH was found for measurements of OH oxidation of ambient Amazon forest air in an OFR. At higher OH exposures in the reactor (>  1  ×  1012 molec cm−3 s), the mass loss of IEPOX-SOA due to heterogeneous reaction was mainly due to revolatilization of fragmented reaction products. We report, for the first time, OH reactive uptake coefficients (γOH =  0.59 ± 0.33 in SE US and γOH =  0.68 ± 0.38 in Amazon) for SOA under ambient conditions. A relative humidity dependence of kOH and γOH was observed, consistent with surface-area-limited OH uptake. No decrease of kOH was observed as OH concentrations increased. These observations of physicochemical properties of IEPOX-SOA can help to constrain OA impact on air quality and climate.

Isoprene-epoxydiols-derived secondary organic aerosol (IEPOX-SOA) can contribute substantially to organic aerosol (OA) concentrations in forested areas under low NO conditions, hence significantly influencing the regional and global OA budgets, accounting, for example, for 16–36 % of the submicron OA in the southeastern United States (SE US) summer. Particle evaporation measurements from a thermodenuder show that the volatility of ambient IEPOX-SOA is lower than that of bulk OA and also much lower than that of known monomer IEPOX-SOA tracer species, indicating that IEPOX-SOA likely exists mostly as oligomers in the aerosol phase. The OH aging process of ambient IEPOX-SOA was investigated with an oxidation flow reactor (OFR). New IEPOX-SOA formation in the reactor was negligible, as the OFR does not accelerate processes such as aerosol uptake and reactions that do not scale with OH. Simulation results indicate that adding 100 µg m-3 of pure H2SO4 to the ambient air allows IEPOX-SOA to be efficiently formed in the reactor. The heterogeneous reaction rate coefficient of ambient IEPOX-SOA with OH radical (kOH) was estimated as 4.0 ± 2.0 ×10-13 cm3 molec-1 s-1, which is equivalent to more than a 2-week lifetime. A similar kOH was found for measurements of OH oxidation of ambient Amazon forest air in an OFR. At higher OH exposures in the reactor (> 1 × 1012 molec cm-3 s), the mass loss of IEPOX-SOA due to heterogeneous reaction was mainly due to revolatilization of fragmented reaction products. We report, for the first time, OH reactive uptake coefficients (γOH = 0.59±0.33 in SE US and γOH = 0.68±0.38 in Amazon) for SOA under ambient conditions. A relative humidity dependence of kOH and γOH was observed, consistent with surface-area-limited OH uptake. No decrease of kOH was observed as OH concentrations increased. These observations of physicochemical properties of IEPOX-SOA can help to constrain OA impact on air quality and climate.