Microplastics are posing an escalating threat to both ecological systems and human health. Yet, current methods for investigating their bioaccumulation are highly invasive, requiring destructive analysis of ex vivo tissues via mass spectrometry, dye labelling, or Raman microspectroscopy. This limits the study of biodistribution dynamics in preclinical models and human populations, leaving an urgent need for non-invasive alternatives. Meeting this challenge, for the first time in living tissue, the native optical absorption properties of microplastics are exploited to generate photoacoustic signal - imageable ultrasound emission following thermalisation of pulsed laser light. Distinct optical absorption profiles enable microplastic differentiation from endogenous biological signal sources, long-term tracking over 2 months in a mouse model, and microscale resolution of particle features verified histologically. This novel approach overcomes previous limitations of optical and nuclear imaging methods relying on fluorescent dyes or radio-isotopes, going beyond small transparent organisms such as zebrafish or nematodes, and half-life-dependent timescales, respectively. By enabling serial monitoring of microplastic biodistribution dynamics, this technique will help interrogate interacting factors such as ingestion route, microplastic shape, size and polymer type - and their effects on accumulation, degradation, clearance, and disease in animal models, and, ultimately, human subjects.

To characterize the limits of detection sensitivity in photoacoustic imaging, we applied ultra-sensitive sensors to measure the "acoustic background" - random pressures arising from thermal vibrations. The results could help guide the design of photoacoustic systems. (c) 2025 The Author(s)

Photoacoustic tomography (PAT) has the potential to become a widely used imaging tool in preclinical studies of small animals. This is because it can provide non-invasive, label free images of whole-body mouse anatomy, in a manner which is challenging for more established imaging modalities. However, existing PAT scanners are limited because they either do not implement a full 3-D tomographic reconstruction using all the recorded photoacoustic (PA) data and/or do not record the available 3-D PA time-series data around the mouse with sufficiently high spatial resolution ( $\sim 100\mu $ m), which compromises image quality in terms of resolution, imaging depth and the introduction of artefacts. In this study, we address these limitations by demonstrating an all-optical, multi-view Fabry-Perot based scanner for whole body small animal imaging. The scanner densely samples the acoustic field with a large number of detection points (>100,000), evenly distributed around the mouse. The locations of the detection points were registered onto a common coordinate system, before a tomographic reconstruction using all the recorded PA time series was implemented. This enabled the acquisition of high resolution, whole-body PAT images of ex-vivo mice, with anatomical features visible across the entire cross section.

Supplementary video showing fluorescence-guided resection of a subcutaneous model of Neuroblastoma injected with anti-GD2 fluorescence probes

In vivo validation of tumor uptake of anti–GD2-IR800 and anti–GD2-IR12 using a commercially available clinical NIR-I imaging device. A, Comparison of fluorescent images of the unexposed tumors at different time points postinjection of either anti–GD2-IR800 or anti–GD2-IR12. The laser boost setting of the EleVision IR Platform was set to arbitrary units for 24, 48, 72, 96 hours and exposed anti–GD2-IR12 images, respectively. B, Bar chart showing MFI of the unexposed tumors post anti–GD2-IR800 and anti–GD2-IR12 injections. Quantification was performed using the EleVision software. C, Bar chart of the TBR. Error bars are calculated from the SD across 5 points manually selected in each ROI. Anti–GD2-IR800, n = 2 at all time points; anti–GD2-IR12, n = 2 at 24 hours, n = 1 at 48, 72, and 96 hours. D, Images showing stages of tumor excision after injection of anti–GD2-IR800 under white light observation and NIR-I fluorescence. A residual tumor (3 × 5 mm) was identified on fluorescence imaging and subsequently excised. E, Histopathologic evaluation of both the main tumor and residual tissue, confirming the presence of viable neuroblastoma.

Biological evaluation of anti–GD2-IR800 and anti–GD2-IR12 using a preclinical NIR-I imaging system in vivo. A, IVIS Spectrum images of the exposed tumors at 24, 48, 72, and 96 hours postinjection of anti–GD2-IR800 (n = 3) and anti–GD2-IR12 (n = 3) in mice bearing subcutaneous LAN-1 neuroblastoma tumors. White dotted lines show the ROIs drawn to quantify tumor to background ratio. B, Graph bar showing fluorescence intensity of the tumor and the background at 24, 48, 72, and 96 hours post conjugates injection. C, Tumor-to-background ratio at 24, 48, 72, and 96 hours after the injection of the conjugates. Error bars represent the SE across individuals. D,Ex vivo fluorescence images of the resected organs. The white dotted lines delineate the regions used to quantify the fluorescence signals. E, MFI for each organ at each time point and for the background card (BG). Fluorescence intensity decreases over time, with the anti–GD2-IR800 being consistently brighter than the anti–GD2-IR12 (MFIIR800/MFIIR12 = 1.90 ± 0.36 for tumor, P = 10−20, three-way ANOVA). Error bars represent the SE across individuals. F, MFI of tumor relative to MFI of each organ at each time point. Data for control mice is shown in green in the graphs B, C, E, and F.

Overview of the in vivo preclinical study. A, Six to 8-week-old athymic nude female mice LAN-1 xenografts were injected with either anti–GD2-IR800 or anti–GD-IR12. The control mice were not injected. Mice were imaged at 24, 48, 72, and 96 hours using the IVIS Spectrum and the multispectral NIR-I/SWIR fluorescence imaging system. B, At each end time point, mice were euthanized and imaged using the IVIS (n = 3) and the multispectral NIR-I/SWIR fluorescence imaging system (n = 1). The unexposed tumors were imaged, followed by the exposed tumors and the excised organs of interest.

Imaging of stained cell pellets beneath tissue-mimicking material using multispectral NIR-I/SWIR fluorescence imaging. A, Microcentrifuges tubes containing pellets of 2 × 106 GD2-positive cells (LAN-1) and GD2-negative cells (SUPT1-WT) stained with 100 nmol/L of anti–GD2-IR800 and covered with a 2% emulsion intralipid. The pellet is located at the tip of the tube. The fluid level is marked in red on the Petri dish. B and C, Multispectral NIR-I/SWIR fluorescence images were captured. Images show the anti–GD2-IR800–stained GD2-positive cells (LAN-1; B) and the anti–GD2-IR800–stained GD2-negative cells (SUPT1-WT; C). White dotted lines show the approximate location of the microcentrifuge tube. D, Line profiles across the fluorescence images were fitted with a Gaussian plus linear background to extract a height and FWHM. Fits shown as a black line. E–G, Bar graphs of the height (E and F) and the FWHM (G and H) versus depth for each wavelength band for GD2-positive (LAN-1) cells stained with anti–GD2-IR800 (E and G) and anti–GD2-IR12 (F and H).

In this work, we present a novel method for characterising the relative variation in hydrophone sensitivity with temperature, addressing a key aspect of measurements in the field of ultrasound metrology. Our study focused on a selection of miniature ultrasonic hydrophones commonly used in medical applications. The method is based on using water as a temperature-sensitive laser-generated ultrasound (LGUS) source for calibration, allowing for flexible characterisation across a wide temperature range. The measurements were performed using both the LGUS method and the established self-reciprocity method. Our results demonstrate good agreement within 5% between the two methods, validating the effectiveness of the LGUS approach. We found that the sensitivity of the tested hydrophones exhibited low temperature dependence less than −0.2% per ∘ C within the studied temperature range from 17 ∘ C up to 50 ∘ C. The presented LGUS method offers greater flexibility than current approaches as it allows for characterisation of membrane hydrophones with small element sizes and non-electrical transducers. By combining the relative sensitivity variation obtained through the LGUS method with the standard calibration at room temperature, absolute values of hydrophone sensitivity can be determined. The expanded uncertainty of our measurements, which was evaluated at temperature intervals of 8 ∘ C, was determined to be on average 10%. Our work provides valuable insights into the temperature dependence of hydrophone sensitivity and lays the foundation for further investigations in this area. The LGUS method holds promise for future enhancements, such as increased bandwidth of the LGUS source and frequency domain analysis, to explore the frequency dependency of sensitivity variation with temperature.

The use of a planar detection geometry in photoacoustic tomography results in the so- called limited-view problem due to the finite extent of the acoustic detection aperture. When images are reconstructed using one-step reconstruction algorithms, image quality is compromised by the presence of streaking artefacts, reduced contrast, image distortion and reduced signal-to-noise ratio. To mitigate this, model-based iterative reconstruction approaches based on least squares minimisation with and without total variation regularization were evaluated using in-silico, experimental phantom, ex vivo and in vivo data. Compared to one-step reconstruction methods, it has been shown that iterative methods provide better image quality in terms of enhanced signal-to-artefact ratio, signal-to-noise ratio, amplitude accuracy and spatial fidelity. For the total variation approaches, the impact of the regularization parameter on image feature scale and amplitude distribution was evaluated. In addition, the extent to which the use of Bregman iterations can compensate for the systematic amplitude bias introduced by total variation was studied. This investigation is expected to inform the practical application of model-based iterative image reconstruction approaches for improving photoacoustic image quality when using finite aperture planar detection geometries.

Semiconducting polymer nanoparticles (SPN), formulated from organic semiconducting polymers and lipids, show promise as exogenous contrast agents for photoacoustic imaging (PAI). To fully realise the potential of this class of nanoparticles for imaging and therapeutic applications, a broad range of active targeting strategies, where ligands specific to receptors on the target cells are displayed on the SPN surface, are urgently needed. In addition, effective strategies for quantifying the level of surface modification are also needed to support development of ligand-targeted SPN. In this paper, we have developed methods to prepare SPN bearing peptides targeted to Epidermal Growth Factor Receptors (EGFR), which are overexpressed at the surface of a wide variety of cancer cell types. In addition to fully characterising these targeted nanoparticles by standard methods (UV-visible, photoacoustic absorption, dynamic light scattering, zeta potential and SEM), we have developed a powerful new NMR method to determine the degree of conjugation and the number of targeting peptides attached to the SPN. Preliminary in vitro experiments with the colorectal cancer cell line LIM1215 indicated that the EGFR-targeting peptide conjugated SPN were either ineffective in delivering the SPN to the cells, or that the targeting peptide itself destabilised the formulation. This in reinforces the need for effective characterisation techniques to measure the surface accessibility of targeting ligands attached to nanoparticles.

Despite aggressive treatments, the prognosis of high-risk NB remains poor. Surgical oncology needs innovative intraoperative devices to help surgeons discriminate malignant tissue from necrotic and surrounding healthy tissues. Changes within the tumor vasculature could be used intraoperatively as a diagnostic tool to guide surgical resection. Here, we retrospectively analyzed the mean vascular density (MVD) of different NB subtypes at diagnosis and after induction chemotherapy using scanned histological samples. One patient was prospectively enrolled, and an ex vivo photoacoustic imaging (PAI) scan was performed on two representative sections to assess its capacity to discriminate different tumor regions. We found that post-chemotherapy, viable areas of differentiating NBs and ganglioneuroblastomas are associated with higher MVD compared to poorly differentiated NBs. Early necrotic regions showed higher MVD than late necrotic and viable regions. Finally, calcified areas showed significantly lower MVD than any other histological component. The acquired PAI images showed a good high-resolution ex vivo 3D delineation of NB margins. Overall, these results suggest that a high-definition preclinical imaging device such as PAI could potentially be exploited to guide surgical resection by identifying different vasculature signatures.

SWIR imaging achieves a higher TBR than NIR-I imaging. A, Normalized band images of exposed tumors at 24 hours post conjugates injection. Images show increased TBR at longer wavelengths. B, Normalized band images (900 and 1,300 nm only) 48, 72, and 96 hours after anti–GD2-IR800 and anti–GD2-IR12 administration. C, TBR versus wavelength band for each conjugate and control mice. Each line represents an individual (one individual per dye per time point). The TBR measured at 1,300 nm 72 hours post anti–GD2-IR12 injection was omitted as an outlier. The upper black line shows the fit of the combined anti–GD2-IR800 and anti–GD2-IR12 data. The lower black line shows the fit of the control data. The gray bands show the 95% confidence interval of the fit (simultaneous functional bounds).

In vitro multispectral NIR-I/SWIR fluorescence imaging of anti–GD2-IR800 and anti–GD2-IR12 A. Schematic representation of selected cell lines (left) and number of cells used (right) to assess the camera sensitivity. Cells were stained with 100 nmol/L of anti–GD2-IR800 and anti–GD2-IR12 and prepared as pellets in microcentrifuge tubes [tubes 1–4, GD2-positive; tubes 5–8, SUPT1-WT (GD2-negative)]. B, Multispectral NIR-I/SWIR images were acquired. Images are shown for 1,050-nm LP filter LAN-1 cells (tubes 1–4) and SUPT1- WT cells (tubes 5–8) with anti–GD2-IR800 and anti–GD2-IR12 on the same fluorescence intensity scale. Yellow dotted lines show the approximate positions of Eppendorf tubes. C, The SBR was calculated as the MFI of each cell pellet divided by the MFI of the control cell pellets for the corresponding cell line and dye. Data are shown for the 850-nm long-pass filter. Error bars are derived from the SEs within the signal and background ROIs. D, MFI of anti–GD2-IR800 and anti–GD2-IR12 decrease towards longer wavelengths. The shaded region represents SE over pixels in ROI. Exposure time = 25 ms.

In vitro validation of anti–GD2-IR800 and anti–GD2-IR12. A, Binding specificity of anti–GD2-IR800 and anti–GD2-IR12 conjugates assessed by flow cytometry in three GD2-positive cell lines (KELLY, LAN-1, and SUPT1-GD2) and in the negative control (SUPT1-WT). B, Both conjugates show higher SBR (median stained fluorescence intensity/median unstained fluorescence intensity) for GD2-positive cell lines than for GD2-negative cell lines. Box represents interquartile range. Bars represent 5% to 95% range. Number of cells = 44,810–70,831. C, Fluorescence and brightfield images of the adherent cells 2 hours after staining with 100 nmol/L of either anti–GD2-IR800 or anti–GD2-IR12 (red). The nuclear counterstain was DAPI (blue). D, Representative section from the Z-stack acquisition of LAN-1 spheroids 24 hours after staining with 100 nmol/L of either anti–GD2-IR800 or anti–GD2-IR12 (red). The nuclear counterstain was DAPI (blue).