Publications

The analysis of vascular morphology and functionality enables the assessment of disease activity and therapeutic effects in various pathologies. Raster-scanning optoacoustic mesoscopy (RSOM) is an imaging modality that enables the visualization of superficial vascular networks in vivo. In murine models of colitis, deep vascular networks in the colon wall can be visualized by transrectal absorber guide raster-scanning optoacoustic mesoscopy (TAG-RSOM). In order to accelerate the implementation of this technology in translational studies of inflammatory bowel disease, an image-processing pipeline for TAG-RSOM data has been developed. Using optoacoustic data from a murine model of chemically-induced colitis, different image segmentation methods are compared for visualization and quantification of deep vascular patterns in terms of vascular network length and complexity, blood volume, and vessel diameter. The presented image-processing pipeline for TAG-RSOM enables label-free in vivo assessment of changes in the vascular network in murine colitis with broad applications for inflammatory bowel disease research.

The development of rapidly distributed and retained probes within the kidneys is important for accurately diagnosing kidney diseases. Although molecular imaging shows the potential for non-intrusively interrogating kidney disease-related biomarkers, the limited kidney contrast of many fluorophores, owing to their relatively low distribution in the kidney, hinders their effectiveness for kidney disease detection. Herein, for the first time, an amino-functionalization strategy is proposed to construct a library of kidney-targeting fluorophores NHcy with tunable emissions from NIR-I to NIR-II. Among these, NHcy-8 is the first small-molecule NIR-II dye without a renal clearance moiety, designed specifically for kidney-targeting imaging. Building on this class of NIR-II fluorophore, the first NIR-II small-molecule kidney-targeting pH probe NIR-II-pH is developed, which exhibits a desirable kidney distribution after intravenous injection and is fluorescent only after activation by acidosis. NIR-II in vivo fluorescence/photoacoustic imaging of kidney disease models induced by cisplatin and renal I/R injury using NIR-II-pH reveals increasingly severe metabolic acidosis as the disease progressed, enabling sensitive detection of the onset of acidosis 36 h (cisplatin group) earlier than clinical methods. Thus, this study introduces a practical NIR-II kidney-targeting probe and provides a useful molecular blueprint for guiding kidney-targeting NIR-II fluorophores as diagnostic aids for kidney diseases.

The optimization of the enzyme-like catalytic selectivity of nanozymes for specific reactive oxygen species (ROS)-related applications is significant, and meanwhile the real-time monitoring of ROS is really crucial for tracking the therapeutic process. Herein, we present a mild oxidation valence-engineering strategy to modulate the valence states of Mo in Pluronic F127-coated MoO3-x nanozymes (denoted as MF-x, x: oxidation time) in a controlled manner aiming to improve their specificity of H2O2-associated catalytic reactions for specific therapy and monitoring of ROS-related diseases. Experimentally, MF-0 (Mo average valence 4.64) and MF-10 (Mo average valence 5.68) exhibit exclusively optimal catalase (CAT)- or peroxidase (POD)-like activity, respectively. Density functional theory (DFT) calculations verify the most favorable reaction path for both MF-0- and MF-10-catalyzed reaction processes based on free energy diagram and electronic structure analysis, disclosing the mechanism of the H2O2 activation pathway on the Mo-based nanozymes. Furthermore, MF-0 poses a strong potential in acute kidney injury (AKI) treatment, achieving excellent therapeutic outcomes in vitro and in vivo. Notably, the ROS-responsive photoacoustic imaging (PAI) signal of MF-0 during treatment guarantees real-time monitoring of the therapeutic effect and post-cure assessment in vivo, providing a highly desirable non-invasive diagnostic approach for ROS-related diseases.

Optoacoustic (photoacoustic) imaging advances allow high-resolution optical imaging much deeper than optical microscopy. However, while label-free optoacoustics have already entered clinical application, biological imaging is in need of ubiquitous optoacoustic labels for use in ways that are similar to how fluorescent proteins propelled optical microscopy. We review photoswitching advances that shine a new light or, in analogy, ‘bring a new sound’ to biological optoacoustic imaging. Based on engineered labels and novel devices, switching uses light or other energy forms and enables signal modulation and synchronous detection for maximizing contrast and detection sensitivity over other optoacoustic labels. Herein, we explain contrast enhancement in the spectral versus temporal domains and review labels and key concepts of switching and their properties to modulate optoacoustic signals. We further outline systems and applications and discuss how switching can enable optoacoustic imaging of cellular or molecular contrast at depths and resolutions beyond those of other optical methods.

NIR-II imaging has the advantages of high sensitivity, spatiotemporal resolution, and high penetration depth, thereby serving as a potential alternative to conventional imaging methods. Herein, a novel NIR-II dye IR-1010 (λex/λem = 1010/1058 nm) is reported with high quantum yield (3.08%) and good stability, by incorporating p-methoxyphenyl groups into a quinolinium cyanine dye. Then a multifunctional nanoprobe, termed IUFP NPs, is developed by the incorporation of upconversion (UC) nanoparticles (NPs), perfluoro-15-crown-5-ether (PFCE), and IR-1010, to display the novel performance of multimodal imaging. Under the single-wavelength excitation (980 nm), IUFP NPs simultaneously emit the NIR-II fluorescence of IR-1010 and visible UC luminescence of UCNPs, and thus realize the UC imaging for cells, and NIR-II fluorescence/photoacoustic/19F magnetic resonance imaging for blood vessels, lymph nodes and tumor in mice. This work affords a novel approach to NIR-II dyes and a general strategy for the design of multimodal imaging probes.

Aristolochic acid I (AAI), a natural compound in aristolochia type Chinese medicinal herb, is generally acknowledged to have nephrotoxicity, which may be associated with mitophagy. Mitophagy is a cellular process with important functions that drive AAI-induced renal injury. Mitochondrial pH is currently measured by fluorescent probes in cell culture, but existing probes do not allow for in situ imaging of AAI-induced mitophagy in vivo. We developed a ratiometric fluorescent/PA dual-modal probe with a silicon rhodamine fluorophore and a pH-sensitive hemicyanine dye covalently linked via a short chain to obtain a FRET type probe. The probe was used to measure AAI-mediated mitochondrial acidification in live cells and in vivo. The Förster resonance energy transfer (FRET)-mediated ratiometric and bimodal method can efficiently eliminate signal variability associated with the commonly used one-emission and single detection mode by ratiometric two channels of the donor and acceptor. The probe has good water-solubility and low molecular weight with two positively charged, facilitating its precise targeting into renal mitochondria, where the fluorescent/PA changes in response to mitochondrial acidification, enabling dynamic and semi-quantitative mapping of subtle changes in mitochondrial pH in AAI-induced nephrotoxicity mouse model for the first time. Also, the joint use of L-carnitine could mitigate the mitophagy in AAI-induced nephrotoxicity.

Objective: The formation of functional vasculature in solid tumours enables delivery of oxygen and nutrients, and is vital for effective treatment with chemotherapeutic agents. Longitudinal characterisation of vascular networks can be enabled using mesoscopic photoacoustic imaging, but requires accurate image co-registration to precisely assess local changes across disease development or in response to therapy. Co-registration in photoacoustic imaging is challenging due to the complex nature of the generated signal, including the sparsity of data, artefacts related to the illumination/detection geometry, scan-to-scan technical variability, and biological variability, such as transient changes in perfusion. To better inform the choice of co-registration algorithms, we compared five open-source methods, in physiological and pathological tissues, with the aim of aligning evolving vascular networks in tumours imaged over growth at different time-points.
Approach:Co-registration techniques were applied to 3D vascular images acquired with photoacoustic mesoscopy from murine ears and breast cancer patient-derived xenografts, at a fixed time-point and longitudinally. Images were pre-processed and segmented using an unsupervised generative adversarial network. To compare co-registration quality in different settings, pairs of fixed and moving intensity images and/or segmentations were fed into five methods split into the following categories: affine intensity-based using 1)mutual information (MI) or 2)normalised cross-correlation (NCC) as optimisation metrics, affine shape-based using 3)NCC applied to distance-transformed segmentations or 4)iterative closest point algorithm, and deformable weakly supervised deep learning-based using 5)LocalNet co-registration. Percent-changes in Dice coefficients, surface distances, MI, structural similarity index measure and target registration errors were evaluated.
Main results:Co-registration using MI or NCC provided similar alignment performance, better than shape-based methods. LocalNet provided accurate co-registration of substructures by optimising subfield deformation throughout the volumes, outperforming other methods, especially in the longitudinal breast cancer xenograft dataset by minimising target registration errors.
Significance:We showed the feasibility of co-registering repeatedly or longitudinally imaged vascular networks in photoacoustic mesoscopy, taking a step towards longitudinal quantitative characterisation of these complex structures. These tools open new outlooks for monitoring tumour angiogenesis at the meso-scale and for quantifying treatment-induced co-localised alterations in the vasculature.

Pompe disease (PD) is a rare autosomal recessive glycogen storage disorder that causes proximal muscle weakness and loss of respiratory function. While enzyme replacement therapy (ERT) is the only effective treatment, biomarkers for disease monitoring are scarce. Following ex vivo biomarker validation in phantom studies, we apply multispectral optoacoustic tomography (MSOT), a laser- and ultrasound-based non-invasive imaging approach, in a clinical trial (NCT05083806) to image the biceps muscles of 10 late-onset PD (LOPD) patients and 10 matched healthy controls. MSOT is compared with muscle magnetic resonance imaging (MRI), ultrasound, spirometry, muscle testing and quality of life scores. Next, results are validated in an independent LOPD patient cohort from a second clinical site. Our study demonstrates that MSOT enables imaging of subcellular disease pathology with increases in glycogen/water, collagen and lipid signals, providing higher sensitivity in detecting muscle degeneration than current methods. This translational approach suggests implementation in the complex care of these rare disease patients.

Metal-modulated croconium dyes with multimodal-imaging and synergistic therapy in the tumor microenvironment have exhibited great potential in tumor theranostics. However, their unideal structure optimization always weakened the efficacy of near-infrared fluorescence-photoacoustic (NIRF/PA) imaging and photothermal therapy (PTT). Here, we screened croconium dye containing two indole groups with better NIRF/PA imaging and PTT in their family, linked to two morpholine rings, and obtained CR-736, as a lysosome-targeting and Fe3+-modulated agent. The established CR-736-Fe3+ nanoplatform was accurately delivered to the breast tumor site, released CR-736 and Fe3+ in the lower acidic lysosome microenvironment, and activated pH-responsive NIRF/PA/magnetic resonance imaging and PTT. Furthermore, ferroptosis generated hydroxyl free radicals and lipid peroxide by consuming GSH and H2O2 by dint of the accumulation of Fe3+ in tumor cells, which resulted in the inhibition of the expression of heat shock proteins and the concomitant recovery of PTT. The synergistic therapy of PTT, ferroptosis, and chemodynamics was further optimized to the maximal extent in tumor lysosome acidic microenvironment and proved both in vitro and a mouse tumor model. This study opens a new avenue in designing excellent and unique croconium-based nanoplatforms, synergizing multiple tumor theranostic methods, and further optimizing the theranostic effects in tumor microenvironment.

Immune checkpoint blockade (ICB) therapy, while showing promise in various cancers, exhibits limited effectiveness in hepatic carcinoma due to the tumor’s immunosuppressive microenvironment (TME) and challenges associated with immune cell infiltration. Efforts to transform the “cold” TME into an “inflamed” state, notably through chemo-immunotherapy, have sparked interest due to their potential to induce immunogenic cell death and augment the infiltration of cytotoxic T lymphocytes (CTLs). Nonetheless, the efficacy of chemo-immunotherapy is often compromised by suboptimal pharmacokinetics, poor tumor accumulation, and off-target toxicity. Herein, in response, we introduce an innovative, milder thermal therapeutic approach leveraging gold nano frameworks with mesopores for the targeted delivery of the immunostimulant imiquimod and NIR-II photothermal therapy. This strategy employs targeted molecule modifications to ensure precise tumor targeting, guided by photoacoustic imaging. Subsequent to mild thermal treatment, there is a release of immunogenic proteins (CRT and HSP90), enhancing tumor immunogenicity. Assisted by imiquimod, substantial CTL infiltration occurs, accompanied by pro-inflammatory factor release (TNF-α, IL-6), transforming M2 macrophages into the M1 phenotype. Ultimately, the proposed strategy combines PD-L1/PD-1 blockade, imiquimod and mild thermal treatment to synergistically enhance tumor immunogenicity, remodel the TME, and restrain hepatic carcinoma, making strides in ICB synergistic immune-thermal therapy.