Photoacoustic imaging is promising for breast cancer diagnosis. However, image quality diminishes with greater melanin content with delay-and-sum (DAS) beamforming, which can be resolved with short-lag spatial coherence (SLSC) beamforming. We investigated the applicability of SLSC beamforming to photoacoustic breast data acquired from 29 patients with skin tone individual typology angles (ITAs) ranging −78° (dark) to 71° (very light), acquired with 757 nm and 1064 nm wavelengths. In shallow targets (<5 mm), with 1064 nm wavelength, target detectability (defined as a scaled average of generalized contrast-to-noise and signal-to-noise ratios) decreased from a mean of 0.80 with DAS beamforming to a mean of 0.33 with SLSC beamforming (p < 0.05). Shallow targets were not visible with 757 nm wavelength when ITA < 14°. In deeper targets, when ITA ≤ 0°, the mean target detectability aggregated across both wavelengths was 0.74 and 0.85 with DAS and SLSC beamforming, respectively. Results were validated with simulated and/or phantom data, and statistical significance was demonstrated with a linear mixed-effects model (p < 0.05), providing the first known tripartite analysis to assess competing interactions among beamformers, wavelengths, and target depths in clinical photoacoustic imaging.
Fracture healing is a dynamic biological process involving inflammation, activation of callus-forming skeletal progenitor cells (CaSPCs), and tissue remodeling, in which redox regulation plays a critical role. However, there is no established imaging technique that evaluates CaSPC-related redox activity at fracture sites in vivo, as conventional imaging modalities reflect structural changes rather than cellular activity. In vivo dynamic nuclear polarization magnetic resonance imaging (DNP-MRI) enables visualization of tissue redox status. In this study, we investigated the utility of DNP-MRI to visualize spatiotemporal redox changes at fracture sites using a mouse tibial fracture model. In vivo DNP-MRI with the nitroxyl radical probe carbamoyl-PROXYL (CmP) revealed stage-dependent, accelerated CmP signal decay within the fracture callus during early- and mid-phase healing. Complementary in vitro redox analyses demonstrated that CaSPCs exhibit significantly higher CmP decay rates than intact bone-derived skeletal progenitor cells (IB-SPCs). Ex vivo analyses revealed that CaSPCs exhibited higher mitochondrial ROS production than IB-SPCs. These findings indicate that redox dynamics within the fracture callus are stage-dependent and reflect intrinsic metabolic features associated with CaSPCs. In vivo DNP-MRI represents a functional imaging approach for visualizing redox metabolism during fracture healing, providing metabolic information beyond structural imaging and enabling earlier evaluation of bone regeneration.
X-ray micro-computed tomography (μCT) is widely used in biomedical research for non-destructive, high-resolution imaging. Synchrotron Radiation Phase-Contrast μCT (SR-PCI-μCT) further enhances image quality with higher Signal to Noise Ratio (SNR), improved contrast, and faster acquisition. However, segmenting SR-PCI-μCT images remains challenging due to their heterogeneous image property and limited training data. Additionally, the increasing throughput of synchrotron facilities demands robust, efficient segmentation methods. This paper reviews a list of recent segmentation approaches in biomedical μCT. Traditional methods remain simple and effective for simple, high-contrast structures but require extensive tuning and generalize poorly to complex, low-contrast soft tissues. Data-driven models provide higher accuracy and robustness yet rely heavily on large expert-annotated datasets, limiting reproducibility and cross-dataset adaptability. Recent advance on vision transformers have shifted the paradigm from task-specified to more domain-specified segmentation, though these techniques are still evolving and require adaptation for SR-PCI-μCT studies. This survey provides the first bi-modality review covering both laboratory and synchrotron biomedical μCT segmentation. It consolidates recent segmentation methodologies, identifies major trends in deep-learning techniques, and highlights current limitations across SR-PCI-μCT. Additionally, it outlines open challenges to guide future research and practical advancements in biomedical μCT segmentation.
Sentinel lymph node (SLN) mapping is essential for cancer staging and treatment planning. Superparamagnetic iron oxides (SPIOs) are used for magnetic SLN localization in breast cancer and are being explored in cancers with complex lymphatic drainage pathways. Magnetic particle imaging (MPI) directly detects SPIOs and has demonstrated feasibility for quantitative lymphography in mouse models. However, most MPI studies have relied on preclinical SPIO formulations not intended for human use. Here, we evaluated three clinically available or investigational SPIOs for MPI, including Magtrace, Resotran, and FerroTrace. SPIOs were characterized in vitro using 2D MPI and relaxometry, benchmarked against the preclinical standard VivoTrax. Magtrace and Resotran demonstrated in vitro performance comparable to VivoTrax, whereas FerroTrace exhibited lower peak signal and resolution by relaxometry. In vivo longitudinal MPI lymphography was performed in healthy C57BL/6 mice following intradermal injection of SPIOs. Magtrace and FerroTrace produced robust lymph node labeling, with MPI signal persisting for at least four weeks across all SPIOs. Feasibility of clinical-scale MPI for complex lymphatic mapping was demonstrated using a human-sized head-and-neck phantom modeling bilateral lymphatic drainage. Together, these results establish Magtrace, Resotran, and FerroTrace as suitable magnetic tracers for MPI lymphography and support their use in future human MPI applications.
We explored the feasibility and preliminary diagnostic capability of ultrasound-guided photoacoustic (US-PA) tomography for breast cancer margin assessment by analysing PA derived lipid, collagen, and hemoglobin distribution patterns. Fifty-three ex-vivo specimens from breast-conserving surgeries were imaged using a handheld US-PA imaging probe illuminated between 700 and 1100 nm across radial margins. Photoacoustic patterns were analysed and assessed for margin involvement using a proprietary algorithm and compared with standard of care against histopathology as the gold standard. US-PA imaging demonstrated 95.45% sensitivity and 76.33% specificity for margin assessment with a positive predictive value (PPV) of 34.43.% and negative predictive value (NPV) of 99.23%. Clinical standard of care showed 40.91% sensitivity, 88.17% specificity, 31.03% PPV and 91.98% NPV. Analysis of false positives revealed that 75% occurred within 5 mm of the margin, with 35% within 2 mm. Overall, US-PA imaging demonstrated superior sensitivity, specificity, PPV, and NPV compared to standard approaches, supporting its potential to reduce missed positive margins. The high false positive rate, primarily due to close margins and tissue compression effects, reflects a conservative classification strategy. Detection of biochemical markers, particularly collagen highlights potential of US-PA in enhancing margin assessment accuracy, although further refinement is needed to improve specificity.
Long-term live-cell fluorescence imaging requires low excitation intensity or short exposure times to reduce phototoxicity, which leads to insufficient signal-to-noise ratios (SNR) in the acquired data. Under such conditions, existing reconstruction methods struggle to balance noise reduction and fine structure preservation, often resulting in distorted reconstructions or severe artifacts. Here, we present HiFi-DeconvFormer, a physics-guided self-supervised reconstruction framework for super-resolution deconvolution imaging. By combining a spatial-redundancy-based training strategy with a network architecture that incorporates physical imaging models, our approach enables robust self-supervised learning without requiring ground truth data. Leveraging windowed Transformers and multi-modal regularization, the framework effectively captures long-range biological continuity and high-frequency edge features, ensuring accurate recovery of weak signals. Extensive benchmarks show that HiFi-DeconvFormer outperforms state-of-the-art methods under low-SNR conditions, achieving high-fidelity, artifact-free reconstruction of dynamic subcellular processes.
Metabolic dysfunction and hepatocellular injury coexist in liver disease but remain difficult to disentangle non-invasively. Hyperpolarized magnetic resonance imaging (HP MRI) enables in vivo metabolic interrogation but has primarily been applied to rapid exchange pathways. Here we evaluate whether complementary hyperpolarized molecular probes provide pathway-specific information on hepatic injury and methyl-donor metabolism in a rat model of steatohepatitis induced by a methionine- and choline-deficient diet. Hyperpolarized [1,4-13C2]fumarate MRI detected robust conversion to [1,4-13C2]malate in diseased livers, reflecting injury-associated fumarate hydratase accessibility following loss of membrane integrity. In the same animals, hyperpolarized 15N,d9-betaine enabled detection of downstream 15N,d6-dimethylglycine as a readout of hepatic one-carbon metabolism over a multi-minute timescale. While malate signal increased markedly in steatohepatitis, dimethylglycine formation was reduced despite comparable parent betaine signal, indicating impaired metabolic engagement rather than altered substrate delivery. These hyperpolarized contrast mechanisms provide complementary metabolic readouts of hepatic injury and methyl-donor metabolism in vivo, extending hyperpolarized MRI toward probing endogenous metabolic processes on extended timescales.
Endometrial cancer is the most common malignancy of the female reproductive tract, yet diagnosis relies on invasive tissue sampling with risk of false negatives. We report an initial investigation of catheter-based 3D optical coherence tomography (OCT) integrated with functional, structural, and radiomic feature analysis for noninvasive endometrial assessment in ex vivo specimens. A custom 3.1-mm OCT catheter acquired volumetric images from 57 intact uterine specimens spanning normal endometrium, benign conditions, high-risk endometrial intraepithelial neoplasia, and endometrial cancer. Functional, structural, and radiomic features were extracted from OCT intensity and scattering images. Statistically significant features were selected to construct a cosine similarity matrix and a cosine-similarity network graph. Cosine-similarity visualization demonstrated exploratory sensitivity and specificity of 94% and 87%, while a cross-validated Logistic Regression Classifier yielded sensitivity and specificity of 91% and 83%. These findings support catheter-based 3D OCT as a promising noninvasive optical biopsy approach to improve detection of endometrial cancer.
Theranostics, which has been built on the deep foundations of diagnostic and therapeutic nuclear medicine laid over the past century, is now emerging as an important pillar of oncological care. Perspectives gained from over 30 years in the field and working in an environment that allowed development of a multidisciplinary team will hopefully guide and inspire the next generation of “theranosticians” to build on these advances.
Ensuring fairness and explainability is essential for the development of ethical, reliable, and effective AI systems in healthcare. Bias in AI models can contribute to disparities in clinical outcomes, challenging equity in medical decision-making. Content-Based Image Retrieval (CBIR) offers interpretable, visual tools to support diagnostic processes; however, these tools remain susceptible to biases inherent in the data. This study investigates covariate bias arising from differences in scanning devices within Foundation Models (FMs) used for CBIR in histopathology. We introduce a unique dataset comprising spatially co-registered images derived from the same histopathology slides scanned using two distinct scanners. This design enables a targeted analysis of scanner-induced variability in FM representations. Among the FMs assessed, Vision Transformer (ViT)-based architectures such as UNI, Virchow2, and GigaPath, demonstrated top performance and best generalization properties across scanners.
Near-infrared (NIR) fluorescence-guided surgery (FGS) is limited by operator-dependent acquisition and non-uniform datasets, hindering quantitative comparison between users, devices, and institutions. To address these limitations, we evaluated an advanced wearable Cancer Vision Goggles (CVG) platform that standardizes imaging via dual green-pointer alignment, enabling reproducible acquisition geometry. The preclinical component benchmarked imaging standardization, quantitative robustness, and agreement with established systems, whereas the clinical arm assessed feasibility and performance relative to an FDA-approved system. Performance was evaluated using quantitative endpoints, including tumor-to-nontumor ratio (TNR), normalized intensity maps (NIMs), and Sørensen-Dice (Dice) coefficient spatial overlap. CVG achieved comparable or superior tumor contrast with high spatial overlap, as confirmed by these quantitative analysis metrics. Unlike handheld systems, CVG maintained stable fluorescence detection with no significant change in tumor-to-nontumor ratio from 10 to 60 cm, enabling reproducible quantitative imaging over a broad working-distance range. Extension to human tumors from patients injected with an NIR molecular probe (ClinicalTrials.gov: NCT05576974, 04/08/2025) demonstrated performance equivalent to that of an established FGS system with a substantial footprint in the operating room. In addition, CVG provided practical advantages through standardized single-operator acquisition, reduced operator-dependent variability relative to handheld or cart-based imaging, and quantitative real-time threshold-based visualization. These findings establish a quantitatively validated wearable platform that standardizes FGS from preclinical benchmarking to clinically relevant tumor assessment.
Immune activity within tumors and secondary lymphoid organs critically influences cancer progression, metastasis, and treatment response. We present a dual-nucleus (1H/19F) molecular MRI platform for non-invasive, high-resolution profiling of the tumor-immune microenvironment in immunocompetent mouse models. Tumor viability was visualized by engineering breast cancer cells to express a novel mouse-derived 1H MRI reporter gene, enabling in vivo differentiation of viable and necrotic tumor regions. Concurrently, 19F MRI using perfluorocarbon (PFC) nanoemulsions enabled longitudinal tracking of immune cell infiltration, extending beyond conventional tumor-associated macrophage-focused approaches. Ex vivo analyses confirmed PFC uptake across diverse immune subsets, with tissue- and context-specific variations in 19F signal driven by differences in cell abundance and labeling efficiency. Notably, 19F MRI revealed a predominantly myeloid signature within tumors, a mixed myeloid/lymphoid profile in the spleen, and a lymphoid-skewed signal in tumor-draining lymph nodes. By integrating tumor-specific 1H imaging with immune-resolving 19F imaging, this single-modality platform offers a comprehensive view of tumor architecture and immune cell presence within tumors. Our imaging approach enables discrimination of immunologically dense tumors and offers critical insights into the interpretation of 19F MRI signals within immune competent animal models, providing an immunoimaging tool that increases the translational relevance of preclinical therapeutic insights.
Altered choline, glutamine, and glucose metabolism form a triumvirate of metabolic reprogramming in most cancers that significantly influences growth, progression, and response to treatment. Photoimmunotherapy (PIT) is a highly target-specific treatment where a targeting antibody (Ab) is conjugated to a photosensitizing dye, IR700, that damages the target only when exposed to near-infrared (NIR) light irradiation. The requirement of an extracellular target has restricted PIT targeting to cell surface receptors and antigens. Here, for the first time, we exploited the extracellular domain of three metabolic transporters, CTL1 for choline, ASCT2 for glutamine, and GLUT1 for glucose for PIT, to demonstrate metabolotheranostics of cancer cells. We analyzed the TCGA database to establish increased expression of the three transporters in human pancreatic ductal adenocarcinoma (PDAC). For the PIT studies, we used two patient-derived PDAC cell lines selected for differences in transporter expression and demonstrated an expression-dependent reduction of cell viability following PIT. A single CTL1-PIT treatment of Pa04C tumors resulted in the eradication of four out of five established tumors. In PDAC, the PIT of metabolic transporters would be most effective in the intraoperative setting, where it could significantly impact cancer cells that may have invaded critical structures.
Cancer immunotherapy improves survival, yet many patients exhibit primary or acquired resistance. We established a combinatorial immunotherapy (COMBO) consisting of tumor antigen-specific Th1 cells and dual immune checkpoint blockade and aimed to noninvasively identify sites of immune activation associated with therapeutic response. Using NF-κBLuc-reporter mice, we longitudinally monitored NF-κB activation by using in vivo bioluminescence imaging in the tumor microenvironment (TME) and bone marrow (BM) of mice bearing OVA-expressing MC38 adenocarcinoma (responder) or B16 melanoma (non-responder). COMBO treatment induced tumor regression in OVA-MC38 but not OVA-B16 tumors. Responsive tumors showed significantly increased NF-κB activation in the TME, whereas resistant melanomas displayed no therapy-induced NF-κB activation. In contrast, BM NF-κB activity was reduced upon COMBO treatment in both models. Immunofluorescence and flow cytometry analyses revealed NF-κB activation in tumor-infiltrating MPO⁺ neutrophils and a concomitant reduction of CD11b⁺Gr-1high neutrophils in the BM in OVA-MC38 bearing mice, suggesting therapy-driven myeloid cell egress. Thus, therapeutic efficacy strongly correlated with NF-κB activation within the TME, while systemic BM changes reflected immune mobilization. Longitudinal imaging of NF-κB activity may enable early discrimination between therapy-sensitive and -resistant tumors in preclinical models.
Altered metabolism and perfusion are hallmarks of numerous diseases, yet simultaneous evaluations in vivo remain limited. We present a dual-purpose hyperpolarized probe combining [1-¹³C]pyruvate and ¹³C-tert-butyl alcohol (13C-TBA) for concurrent metabolic and perfusion imaging. An optimized formulation produced high polarizations with short build-up times. In vivo imaging in healthy and tumor-bearing rats demonstrated robust signals of TBA and pyruvate metabolism. This co-polarized dual probe offers strong preclinical and translational potential.
Light sheet fluorescence microscopy (LSFM) is increasingly appreciated as the gold standard for gentle, volumetric imaging with fast acquisition speeds and/or long imaging durations. However, the often-constrained sample space of these microscopes has precluded a specific class of biological specimens from being studied with these tools: those requiring an air-liquid interface (ALI). Here, we present a device for robust imaging at ALI on an upright light sheet microscope with dipping objectives. We demonstrate the system using three relevant use-cases: ex vivo embryonic mouse salivary glands, human epidermal equivalent cultures, and in vivo adult Drosophila melanogaster brains. While the device presented is engineered for one specific light sheet microscope design, it provides a blueprint for easy adaptation to other systems. In doing so, it can potentially spur the use of LSFM for model systems that have so far been unable to take advantage of this powerful technology.
Immunotherapies such as checkpoint inhibitors (i.e. anti-PD-1) and peptide-based therapies (DPX-Survivac) have strong potential for treatment of epithelial ovarian cancer, the most lethal gynecological malignancy. Magnetic resonance imaging (MRI) can be used to track tumor growth and iron-labeled immune cells longitudinally at the individual level. We studied MRI immune cell tracking in a murine model of ovarian cancer using a clinically relevant treatment combination of DPX-Survivac, anti-PD-1, and an intermittent low dose of Cyclophosphamide (CPA). HHD-DR1 mice were orthotopically implanted with mouse ovarian surface epithelial (MOSE) cancer cells. Myeloid and CD8+ cells were isolated from matched donor mice, labeled with superparamagnetic iron oxide (SPIO) and were scanned using MRI on days 42, 49 and 56. Tumor volumes in the treatment group as measured by MRI were significantly lower than in the control group (p < 0.01). The density of SPIO-labeled myeloid and CD8+ T cells in tumors was higher in the treatment group than in the control group. This study provides insights into how MRI can be used in concert with biological assays to study how immunotherapy and chemotherapy combinations exert their antitumor effects.
Abstract The radiohybrid (rh) design of radiopharmaceuticals has recently produced new theranostics suitable for both positron emission tomography (PET) imaging and peptide receptor radionuclide therapy (PRRT). This approach aims to address the limitations of current medical radionuclides by offering a new strategy for combining radionuclides that previously lacked both therapeutic and diagnostic applications. Here, we report on a somatostatin receptor subtype 2 (sstR2)-targeted radiohybrid compound, rhTATE4, which features a bifunctional silicon-based fluoride acceptor (SiFA) - named (SiFA)SeFe - for 18F-labeling, along with a DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelator for 177Lu-coordination. The rh-theranostic agent demonstrates similar in vitro behavior compared to the gold standards [177Lu]Lu-DOTA-TATE and SiFAlin-TATE, along with an exceptionally high tumor uptake (53.58 ± 5.51% ID/g for the radiofluorinated version) after 1 h post-injection in AR42J tumor-bearing mice, making it ideal for imaging. Moreover, clearance from normal tissues and considerable tumor retention (10.32 ± 7.04%ID/g) for [177Lu]Lu-TATE4 were observed at 24 p.i., suggesting good therapeutic applicability.
Extracellular arterial activity of the pro-inflammatory enzyme myeloperoxidase (MPO) destabilizes atherosclerotic plaque and associates with future atherothrombosis. To facilitate first-in-human studies using extracellular MPO activity as a molecular imaging target to identify high-risk atherosclerotic plaque, we describe [68Ga]Ga-IEMA, a NODAGA-based positron emission tomography (PET) radiotracer that provides an index for extracellular MPO activity. Synthesis of [68Ga]Ga-IEMA was achieved in five steps and with high radiolabelling efficiency. [68Ga]Ga-IEMA self-oligomerized and bound to proteins upon exposure to enzymatically active MPO, did not cross-cell membranes and was stable in human serum in vitro, while [68Ga]Ga-IEMA had favorable blood kinetics and stability in circulation in vivo. [68Ga]Ga-IEMA PET imaging in a mouse model of plaque instability revealed enhanced signal in unstable compared with stable plaque and plaque-free arteries. These data indicate that [68Ga]Ga-IEMA is a promising translational candidate for the non-invasive identification of high-risk atherosclerotic plaques and the evaluation of therapies targeting arterial inflammation.
