The editorial highlights articles in a JBO special section, as well as emerging trends in small-animal molecular imaging.

.SignificanceHyperspectral imaging (HSI) is an advanced spectral imaging technique that captures spatial and spectral information across numerous wavelength bands. This capability allows tissue characterization, disease detection and diagnosis, surgical guidance, and digital histopathology, making it an increasingly valuable tool with wide biological and medical applications.AimWe aim to provide readers with (1) an understanding of the principles and technological advancements in HSI, (2) a comprehensive overview of HSI data processing and analysis methods, and (3) an updated survey of biomedical applications, from disease detection, intraoperative imaging, to histopathology.ApproachA systematic literature search was conducted using PubMed and Google Scholar with the keyword “hyperspectral imaging.” We previously published a comprehensive review paper on medical HSI in 2014, which was widely cited in the field. Therefore, this updated review focused on new technology advancements and emerging applications. Based on their biological and medical relevance, 612 HSI papers were included and analyzed in this review.ResultsRecent advances in HSI span both hardware and computational techniques, including improvements in sensor technology, data processing and analysis, short-wave near-infrared imaging, and deep-learning and AI tools. HSI is actively explored for various applications in oncology, neurology, ophthalmology, dermatology, cardiology, gastroenterology, hepatology, wound care, endocrinology, dentistry, infectious disease, plastic and reconstructive surgery, general surgery, intraoperative guidance, histopathology, microbiology, nanopathology, and pharmacology.ConclusionsHSI has become an emerging imaging modality in biomedical research and clinical settings. Continued advancements in hardware miniaturization, computational efficiency, and clinical validation will further solidify the role of next-generation HSI in biomedicine.

Craniofacial implants are prone to skin necrosis and exposure, likely due to implant-induced stress and ischemia. However, this relationship has not been quantitatively validated. Identifying an imaging biomarker of skin vascular health could confirm this mechanism and help predict skin failure to mitigate implant complications. Photoacoustic tomography (PAT) was used to monitor changes in the skin vasculature surrounding subcutaneous implants with the goal of obtaining a quantitative metric predictive of implant exposure. Three designs of 3D-printed porous polycaprolactone (PCL) constructs-unimodal block, bimodal block, and unimodal dome-were implanted in 16 hairless mice. PAT was performed biweekly for 16 weeks, and a skeletonization algorithm was applied to quantify vascular density in skin overlying the implants. Mice that developed implant exposure ( ) exhibited a progressive decline in vascular density beginning 6 weeks before visible exposure, whereas nonexposed mice ( ) remained stable. Group differences were significant 4 weeks ( ) and 2 weeks ( ) before exposure onset. These findings establish a quantitative temporal relationship between vascular ischemia and implant exposure. PAT-derived vascular density serves as a predictive biomarker of skin failure, which can be used to enable interventional treatment and improve implant designs.

.SignificanceFreehand optical ultrasound (OpUS) is an emerging imaging modality that utilizes arrays of fiber-optic, photoacoustical ultrasound sources, and a single fiber-optic detector to perform pulse-echo ultrasound imaging in real time and at video rate. The freehand OpUS imaging probes presented to date have utilized either flat-cleaved optical fibers to generate an array of small, circular sources, or featured an additional array of eccentric optical waveguides to generate elliptical sources that improve ultrasound directivity. However, the incorporation of waveguides imposed severe restrictions on the size of the sources, the source pitch within the array, and the overall size of the imaging probe, and ultimately limited achievable image quality and clinical utility of freehand OpUS probes.AimWe present an alternative method for generating eccentric fiber-optic OpUS sources, which incorporates an anisotropic holographic diffuser element (HDE) to shape the profile of fiber-delivered excitation light.ApproachA commercially available HDE was selected and imprinted into a UV-curable adhesive to improve optical resilience. The monolithic and near-uniform structure of the resulting adhesive-imprinted HDE structure greatly facilitated alignment with, and allowed for arbitrary positioning of, arrays of optical fibers, without adding significant bulk.ResultsThe use of an HDE enabled dense, cladding-to-cladding packing of optical fibers, resulting in the first freehand OpUS probe featuring an acoustic source pitch below the spatial Nyquist limit (thus eliminating grating lobe artifacts), high channel count (144 sources), and endoluminal-scale physical dimensions (diameter: 18.5 mm).ConclusionThis HDE-enabled freehand OpUS imaging probe achieved video-rate, real-time imaging at increased image quality and depth and allowed for the first endoluminal freehand OpUS imaging within a realistic imaging phantom.

.SignificancePhotodynamic therapy (PDT) for the treatment of oral cancers and oral potentially malignant lesions can be enhanced by the capability of the photosensitizer to serve as a fluorescence contrast agent for treatment guidance. The development of image-based dosimetry reporters can inform treatment progress in real time to avoid under-treatment, leading to incomplete response and recurrence.AimWe investigate the hypothesis that imaging of protoporphyrin IX (PpIX) photoproduct (PP) accumulation may be leveraged as an implicit PDT dosimetry reporter for PDT using 5-aminolevulinic acid (ALA)-induced PpIX photosensitization.ApproachIn initial spectroscopy studies, we investigate dose-dependent changes in absorption and fluorescence spectra of PpIX corresponding to PP accumulation during red light (635 nm) delivery. We use spectral analysis to select fluorescence excitation and spectral filtering components for PP imaging during treatment. We evaluate the capability for imaging PP accumulation concomitant with PpIX photobleaching in tissue phantoms, 3D oral squamous cell carcinoma (OSCC) models, and in murine xenografts.ResultsSpectroscopy shows fluence-dependent changes in PpIX optical properties, and that excitation of photobleached PpIX with 450 nm light produces fluorescence emission associated with PpIX PPs. An existing handheld intraoral probe is shown to be capable of imaging dose-dependent PP accumulation with the addition of a spectral filter to isolate fluorescence emission longer than 650 nm. PP signal increases concomitant with PpIX photobleaching in a fluence-dependent manner and correlates with the extent of cytotoxic response in 3D cultures. PP accumulation is also observed to occur concomitantly with photobleaching in OSCC subcutaneous xenografts.ConclusionsOverall, the results show that imaging of PP accumulation is feasible by adapting traditional photodiagnosis optical components and may serve as a dosimetry reporter for ALA-PDT, which is complementary to the measurement of PpIX photobleaching.

.SignificancePhotoacoustic tomography (PAT) is an emerging biomedical imaging technology that offers high contrast and high resolution, showing great potential for applications in medical imaging. However, existing regularization methods often lead to instability and artifacts in the reconstruction due to imbalanced regularization parameter settings. To address these issues, we propose a reconstruction algorithm based on the L-alternating direction method of multipliers (ADMM) for PAT, which significantly improves image reconstruction quality and has high clinical application potential.AimWe introduce a nonconvex L1–L2 norm into the variational model and employ the ADMM to decompose the optimization problem into efficiently solvable subproblems. A preconditioned conjugate gradient (PCG) method is further integrated to accelerate the solution of linear systems, thereby improving both reconstruction accuracy and computational efficiency.ApproachWe propose an L-ADMM framework with adaptive weighted L1–L2 regularization for PAT reconstruction. The method employs ADMM to split the optimization into tractable subproblems and uses PCG to efficiently solve linear systems. It achieves stable, high-quality reconstruction under sparse sampling by enhancing sparsity while preserving structural details.ResultsExperiments on vascular and breast models demonstrate that, even with only 64 transducers under sparse sampling, the proposed L-ADMM method achieves peak signal-to-noise ratio values of 37.24 and 36.26 dB and structural similarity index measure values of 0.9766 and 0.9665, respectively. Compared with L2, L1 + L2, L1–L2, TV regularization, and U-Net methods, the proposed algorithm substantially improves image quality, highlighting its feasibility for cost-effective clinical PAT.ConclusionsThe proposed L-ADMM-based reconstruction algorithm, by integrating adaptive regularization with efficient optimization, significantly improves PAT image quality under sparse sampling conditions, offering a feasible solution with strong potential for clinical translation.

.SignificanceIn vivo optical coherence tomography (OCT) of the mouse vitreoretinal vasculature in full depth is technically challenging. Conventional OCT techniques employ axial confocal gating, which induces signal drop-off and limits spatial resolution outside the Rayleigh range.AimOur aim is to develop a multifocal OCT imaging approach using a tunable lens and a registration method that allows the generation of a composite image of the vitreoretinal vasculature while preserving high and uniform lateral spatial resolution, signal intensity, and image contrast in full depth.ApproachA calibration target was developed to characterize the multifocal optical system and quantify the signal intensity, contrast, and resolution. These optical specifications were used to image mice at postnatal day 14. Intra- and inter-volume registration methods were necessary to correct for motion and generate a composite image from single-focus images using weighted averaging.ResultsIn the calibration target, signal intensity and contrast were 20 dB higher in the composite compared with single-focus images. Lateral resolution remained uniform (4 to ). In animals, signal intensity and contrast were 10 to 15 dB higher in the composite compared with single-focus images and highest in the hyaloid vasculature.ConclusionsThis technique is promising in studying the mouse vitreoretinal vasculature during eye development and disease.

.SignificanceMyocardial oxygen metabolism is a key focus of cardiac surgery. It serves as important evidence for surgeons to evaluate surgical quality and surgical plans. However, current clinical methods lack the capability to directly monitor dynamic changes in myocardial metabolism during surgery. Photoacoustic imaging (PAI), a biomedical optical imaging modality, offers real-time assessment of blood oxygen saturation. By visualizing oxygen saturation levels in both blood and muscle tissue, PAI provides a means to infer myocardial metabolic status intraoperatively.AimWe use PAI to observe the differences between infarcted myocardium and normal cardiac muscle and to explore the feasibility of using PAI to monitor myocardial metabolism levels during cardiac surgery.ApproachTen rabbits were randomly divided into experimental and control groups. The animals in the experimental group underwent thoracotomy followed by left anterior descending coronary artery ligation, whereas those in the control group received thoracotomy only. PAI was performed both at the beginning and before the end of the surgical procedure. The PAI results were compared between the two groups to analyze the relationship between myocardial PAI signal changes and oxygen metabolism levels.ResultsFollowing coronary ligation, the experimental group exhibited significant ST-segment elevation on electrocardiography, whereas no notable changes were observed in controls. PAI demonstrated: baseline myocardial oxygen saturation () ranged from 45% to 72% across all rabbits. Ligation-induced ischemia sharply reduced to 1% to 19% in experimental animals. Control animals maintained stable levels throughout the procedure. Histopathological examination confirmed extensive myocardial necrosis in the apical region of ligated rabbits, consistent with the observed functional and metabolic alterations.ConclusionsPAI can detect myocardial oxygen saturation in real-time during surgery and determine the occurrence of myocardial ischemia and changes in oxygen metabolism levels based on differences in oxygen saturation.

.SignificanceLumen segmentation in intravascular optical coherence tomography (IVOCT) images is essential for quantifying vascular stenosis severity, location, and length. Current methods relying on manual parameter tuning or single-frame spatial features struggle with image artifacts, limiting clinical utility.AimWe aim to develop a temporal residual U-Net (TR-Unet) leveraging spatiotemporal feature fusion for robust IVOCT lumen segmentation, particularly in artifact-corrupted images.ApproachWe integrate convolutional long short-term memory networks to capture vascular morphology evolution across pullback sequences, enhanced ResUnet for spatial feature extraction, and coordinate attention mechanisms for adaptive spatiotemporal fusion.ResultsBy processing 2451 clinical images, the proposed TR-Unet model shows a well performance as Dice coefficient = 98.54%, Jaccard similarity (JS) = 97.17%, and recall = 98.26%. Evaluations on severely blood artifact-corrupted images reveal improvements of 3.01% (Dice), 1.3% (ACC), 5.24% (JS), 2.15% (recall), and 2.06% (precision) over competing methods.ConclusionsTR-Unet establishes a robust and effective spatiotemporal fusion paradigm for IVOCT segmentation, demonstrating significant robustness to artifacts and providing architectural insights for temporal modeling optimization.

.SignificancePhotodynamic therapy (PDT) agents activated by near-infrared (NIR) light have demonstrated effectiveness in animal studies. However, clinical trials in humans are lacking due to biocompatibility concerns. We evaluate the feasibility of NIR-PDT using newly developed upconversion nanoparticles–quantum dots–Rose Bengal (UCQRs) through Monte Carlo simulations.AimSurgery, the primary treatment mode for breast cancer, often reduces the quality of life due to scarring, necessitating a less invasive alternative. Herein, we propose an NIR-PDT approach using UCQRs to treat patients with early-stage breast cancer. The treatment can be performed on patients in the prone position using light irradiation alone, significantly reducing the burden on patients. In NIR-PDT using UCQR, a treatment depth of 3 to 4 cm can be expected based on the penetration depth of the 808-nm excitation light.ApproachWe created 150 digital breast phantoms by reconstructing breast slice images from breast computed tomography scans. These phantoms were classified by breast density and tumor depth, and simulations were performed on representative models. The therapeutic effect of NIR-PDT was assessed based on the amount of singlet oxygen generated, calculated from the fluence in the tumor voxels.ResultsThe simulations indicated that tumor depth had a greater impact on the therapeutic outcomes compared with breast contour or structure. In all phantoms where tumors with a 7-mm diameter were embedded at depths of 15 to 25 mm, the generated singlet oxygen exceeded the cell death threshold across all tumor voxels. Shallow tumors between 15 and 20 mm can be treated with 15 or fewer irradiations, whereas deep tumors between 20 and 25 mm are estimated to require up to 45 irradiations.ConclusionsThis virtual clinical trial using 150 digital phantoms suggests that NIR-PDT with UCQRs offers a promising, minimally invasive alternative for treating breast cancer.