First Superferromagnetic Remanence Characterization and Scan Optimization for Super-Resolution Magnetic Particle Imaging.

Magnetic particle imaging (MPI) is a sensitive, high-contrast tracer modality that images superparamagnetic iron oxide nanoparticles, enabling radiation-free theranostic imaging. MPI resolution is currently limited by scanner and particle constraints. Recent tracers have experimentally shown 10× resolution and signal improvements with dramatically sharper M-H curves. Experiments show a dependence on interparticle interactions, conforming to literature definitions of superferromagnetism. We thus call our tracers superferromagnetic iron oxide nanoparticles (SFMIOs). While SFMIOs provide excellent signal and resolution, they exhibit hysteresis with non-negligible remanence and coercivity. We provide the first quantitative measurements of SFMIO remanence decay and reformation using a novel multiecho pulse sequence. We characterize MPI scanning with remanence decay and coercivity and describe an SNR-optimized pulse sequence for SFMIOs under human electromagnetic safety limitations. The resolution from SFMIOs could enable clinical MPI with 10× reduced scanner selection fields, reducing hardware costs by up to 100×.

Superferromagnetic Nanoparticles Enable Order-of-Magnitude Resolution & Sensitivity Gain in Magnetic Particle Imaging.

Magnetic nanoparticles have many advantages in medicine such as their use in non-invasive imaging as a Magnetic Particle Imaging (MPI) tracer or Magnetic Resonance Imaging contrast agent, the ability to be externally shifted or actuated and externally excited to generate heat or release drugs for therapy. Existing nanoparticles have a gentle sigmoidal magnetization response that limits resolution and sensitivity. Here it is shown that superferromagnetic iron oxide nanoparticle chains (SFMIOs) achieve an ideal step-like magnetization response to improve both image resolution & SNR by more than tenfold over conventional MPI. The underlying mechanism relies on dynamic magnetization with square-like hysteresis loops in response to 20 kHz, 15 kAm-1 MPI excitation, with nanoparticles assembling into a chain under an applied magnetic field. Experimental data shows a "1D avalanche" dipole reversal of every nanoparticle in the chain when the applied field overcomes the dynamic coercive threshold of dipole-dipole fields from adjacent nanoparticles in the chain. Intense inductive signal is produced from this event resulting in a sharp signal peak. Novel MPI imaging strategies are demonstrated to harness this behavior towards order-of-magnitude medical image improvements. SFMIOs can provide a breakthrough in noninvasive imaging of cancer, pulmonary embolism, gastrointestinal bleeds, stroke, and inflammation imaging.

Magnetic Particle Imaging: An Emerging Modality with Prospects in Diagnosis, Targeting and Therapy of Cancer.

BACKGROUND: Magnetic Particle Imaging (MPI) is an emerging imaging modality for quantitative direct imaging of superparamagnetic iron oxide nanoparticles (SPION or SPIO). With different physics from MRI, MPI benefits from ideal image contrast with zero background tissue signal. This enables clear visualization of cancer with image characteristics similar to PET or SPECT, but using radiation-free magnetic nanoparticles instead, with infinite-duration reporter persistence in vivo. MPI for cancer imaging: demonstrated months of quantitative imaging of the cancer-related immune response with in situ SPION-labelling of immune cells (e.g., neutrophils, CAR T-cells). Because MPI suffers absolutely no susceptibility artifacts in the lung, immuno-MPI could soon provide completely noninvasive early-stage diagnosis and treatment monitoring of lung cancers. MPI for magnetic steering: MPI gradients are ~150 × stronger than MRI, enabling remote magnetic steering of magneto-aerosol, nanoparticles, and catheter tips, enhancing therapeutic delivery by magnetic means. MPI for precision therapy: gradients enable focusing of magnetic hyperthermia and magnetic-actuated drug release with up to 2 mm precision. The extent of drug release from the magnetic nanocarrier can be quantitatively monitored by MPI of SPION's MPS spectral changes within the nanocarrier. CONCLUSION: MPI is a promising new magnetic modality spanning cancer imaging to guided-therapy.

Non-radioactive and sensitive tracking of neutrophils towards inflammation using antibody functionalized magnetic particle imaging tracers.

White blood cells (WBCs) are a key component of the mammalian immune system and play an essential role in surveillance, defense, and adaptation against foreign pathogens. Apart from their roles in the active combat of infection and the development of adaptive immunity, immune cells are also involved in tumor development and metastasis. Antibody-based therapeutics have been developed to regulate (i.e. selectively activate or inhibit immune function) and harness immune cells to fight malignancy. Alternatively, non-invasive tracking of WBC distribution can diagnose inflammation, infection, fevers of unknown origin (FUOs), and cancer. Magnetic Particle Imaging (MPI) is a non-invasive, non-radioactive, and sensitive medical imaging technique that uses safe superparamagnetic iron oxide nanoparticles (SPIOs) as tracers. MPI has previously been shown to track therapeutic stem cells for over 87 days with a ~200 cell detection limit. In the current work, we utilized antibody-conjugated SPIOs specific to neutrophils for in situ labeling, and non-invasive and radiation-free tracking of these inflammatory cells to sites of infection and inflammation in an in vivo murine model of lipopolysaccharide-induced myositis. MPI showed sensitive detection of inflammation with a contrast-to-noise ratio of ~8-13.

Gadolinium-based bimodal probes to enhance T1-Weighted magnetic resonance/optical imaging.

Gd3+-based contrast agents have been extensively used for signal enhancement of T1-weighted magnetic resonance imaging (MRI) due to the large magnetic moment and long electron spin relaxation time of the paramagnetic Gd3+ ion. The key requisites for the development of Gd3+-based contrast agents are their relaxivities and stabilities which can be achieved by chemical modifications. These modifications include coordinating Gd3+ with a chelator such as diethylenetriamine pentaacetic acid (DTPA) or 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), encapsulating Gd3+ in nanoparticles, conjugation to biomacromolecules such as polymer micelles and liposomes, or non-covalent binding to plasma proteins. In order to have a coherent diagnostic and therapeutic approach and to understand diseases better, the combination of MRI and optical imaging (OI) techniques into one technique entity has been developed to overcome the conventional boundaries of either imaging modality used alone through bringing the excellent spatial resolution of MRI and high sensitivity of OI into full play. Novel MRI and OI bimodal probes have been extensively studied in this regard. This review is an attempt to shed some light on the bimodal imaging probes by summarizing all recent noteworthy publications involving Gd3+ containing MR-optical imaging probes. The several key elements such as novel synthetic strategy, high sensitivity, biocompatibility, and targeting of the probes are highlighted in the review. STATEMENT OF SIGNIFICANCE: The present article aims at giving an overview of the existing bimodal MRI and OI imaging probes. The review structured as a series of examples of paramagnetic Gd3+ ions, either as ions in the crystalline structure of inorganic materials or chelates for contrast enhancement in MRI, while they are used as optical imaging probes in different modes. The comprehensive review focusing on the synthetic strategies, characterizations and properties of these bimodal imaging probes will be helpful in a way to prepare related work.

Using magnetic particle imaging systems to localize and guide magnetic hyperthermia treatment: tracers, hardware, and future medical applications.

Magnetic fluid hyperthermia (MFH) treatment makes use of a suspension of superparamagnetic iron oxide nanoparticles, administered systemically or locally, in combination with an externally applied alternating magnetic field, to ablate target tissue by generating heat through a process called induction. The heat generated above the mammalian euthermic temperature of 37°C induces apoptotic cell death and/or enhances the susceptibility of the target tissue to other therapies such as radiation and chemotherapy. While most hyperthermia techniques currently in development are targeted towards cancer treatment, hyperthermia is also used to treat restenosis, to remove plaques, to ablate nerves and to alleviate pain by increasing regional blood flow. While RF hyperthermia can be directed invasively towards the site of treatment, non-invasive localization of heat through induction is challenging. In this review, we discuss recent progress in the field of RF magnetic fluid hyperthermia and introduce a new diagnostic imaging modality called magnetic particle imaging that allows for a focused theranostic approach encompassing treatment planning, treatment monitoring and spatially localized inductive heating.

Combining magnetic particle imaging and magnetic fluid hyperthermia for localized and image-guided treatment.

Magnetic fluid hyperthermia (MFH) has been widely investigated as a treatment tool for cancer and other diseases. However, focusing traditional MFH to a tumor deep in the body is not feasible because the in vivo wavelength of 300 kHz very low frequency (VLF) excitation fields is longer than 100 m. Recently we demonstrated that millimeter-precision localized heating can be achieved by combining magnetic particle imaging (MPI) with MFH. In principle, real-time MPI imaging can also guide the location and dosing of MFH treatments. Hence, the combination of MPI imaging plus real time localized MPI-MFH could soon permit closed-loop high-resolution hyperthermia treatment. In this review, we will discuss the fundamentals of localized MFH (e.g. physics and biosafety limitations), hardware implementation, MPI real-time guidance, and new research directions on MPI-MFH. We will also discuss how the scale up to human-sized MPI-MFH scanners could proceed.

Optimization of Drive Parameters for Resolution, Sensitivity and Safety in Magnetic Particle Imaging.

Magnetic Particle Imaging is an emerging tracer imaging modality with zero background signal and zero ionizing radiation, high contrast and high sensitivity with quantitative images. While there is recent work showing that the low amplitude or low frequency drive parameters can improve MPI's spatial resolution by mitigating relaxation losses, the concomitant decrease of the MPI's tracer sensitivity due to the lower drive slew rates was not fully addressed. There has yet to be a wide parameter space, multi-objective optimization of MPI drive parameters for high resolution, high sensitivity and safety. In a large-scale study, we experimentally test 5 different nanoparticles ranging from multi to single-core across 18.5 nm to 32.1 nm core sizes and across an expansive drive parameter range of 0.4 - 416 kHz and 0.5 - 40 mT/ µ0 to assess spatial resolution, SNR, and safety. In addition, we analyze how drive-parameter-dependent shifts in harmonic signal energy away and towards the discarded first harmonic affect effective SNR in this optimization study. The results show that when optimizing for all four factors of resolution, SNR, discarded-harmonic-energy and safety, the overall trends are no longer monotonic and clear optimal points emerge. We present drive parameters different from conventional preclinical MPI showing ~ 2-fold improvement in spatial resolution while remaining within safety limits and addressing sensitivity by minimizing the typical SNR loss involved. Finally, validation of the optimization results with 2D images of phantoms was performed.

Pulsed Excitation in Magnetic Particle Imaging.

Magnetic particle imaging (MPI) is a promising new tracer-based imaging modality. The steady-state, nonlinear magnetization physics most fundamental to MPI typically predicts improving resolution with increasing tracer magnetic core size. For larger tracers, and given typical excitation slew rates, this steady-state prediction is compromised by dynamic processes that induce a significant secondary blur and prevent us from achieving high resolution using larger tracers. Here, we propose a new method of excitation and signal encoding in MPI we call pulsed MPI to overcome this phenomenon. Pulsed MPI allows us to directly encode the steady-state magnetic physics into the time-domain signal. This in turn gives rise to a simple reconstruction algorithm to obtain images free of secondary relaxation-induced blur. Here, we provide a detailed description of our approach in 1D, discuss how it compares with alternative approaches, and show experimental data demonstrating better than 500- [Formula: see text] resolution (at 7 T/m) with large tracers. Finally, we show experimental images from a 2D implementation.

Noninvasive Anatomical and Functional Imaging of Orthotopic Glioblastoma Development and Therapy using Multispectral Optoacoustic Tomography.

PURPOSE: Here we demonstrate the potential of multispectral optoacoustic tomography (MSOT), a new non-invasive structural and functional imaging modality, to track the growth and changes in blood oxygen saturation (sO2) in orthotopic glioblastoma (GBMs) and the surrounding brain tissues upon administration of a vascular disruptive agent (VDA). METHODS: Nude mice injected with U87MG tumor cells were longitudinally monitored for the development of orthotopic GBMs up to 15 days and observed for changes in sO2 upon administration of combretastatin A4 phosphate (CA4P, 30 mg/kg), an FDA approved VDA for treating solid tumors. We employed a newly-developed non-negative constrained approach for combined MSOT image reconstruction and unmixing in order to quantitatively map sO2 in whole mouse brains. RESULTS: Upon longitudinal monitoring, tumors could be detected in mouse brains using single-wavelength data as early as 6 days post tumor cell inoculation. Fifteen days post-inoculation, tumors had higher sO2 of 63 ± 11% (n = 5, P < .05) against 48 ± 7% in the corresponding contralateral brain, indicating their hyperoxic status. In a different set of animals, 42 days post-inoculation, tumors had lower sO2 of 42 ± 5% against 49 ± 4% (n = 3, P < .05) in the contralateral side, indicating their hypoxic status. Upon CA4P administration, sO2 in 15 days post-inoculation tumors dropped from 61 ± 9% to 36 ± 1% (n = 4, P < .01) within one hour, then reverted to pre CA4P treatment values (63 ± 6%) and remained constant until the last observation time point of 6 hours. CONCLUSION: With the help of advanced post processing algorithms, MSOT was capable of monitoring the tumor growth and assessing hemodynamic changes upon administration of VDAs in orthotopic GBMs.

In vivo tracking and quantification of inhaled aerosol using magnetic particle imaging towards inhaled therapeutic monitoring.

Pulmonary delivery of therapeutics is attractive due to rapid absorption and non-invasiveness but it is challenging to monitor and quantify the delivered aerosol or powder. Currently, single-photon emission computed tomography (SPECT) is used but requires inhalation of radioactive labels that typically have to be synthesized and attached by hot chemistry techniques just prior to every scan. Methods: In this work, we demonstrate that superparamagnetic iron oxide nanoparticles (SPIONs) can be used to label and track aerosols in vivo with high sensitivity using an emerging medical imaging technique known as magnetic particle imaging (MPI). We perform proof-of-concept experiments with SPIONs for various lung applications such as evaluation of efficiency and uniformity of aerosol delivery, tracking of the initial aerosolized therapeutic deposition in vivo, and finally, sensitive visualization of the entire mucociliary clearance pathway from the lung up to the epiglottis and down the gastrointestinal tract to be excreted. Results: Imaging of SPIONs in the lung has previously been limited by difficulty of lung imaging with magnetic resonance imaging (MRI). In our results, MPI enabled SPION lung imaging with high sensitivity, and a key implication is the potential combination with magnetic actuation or hyperthermia for MPI-guided therapy in the lung with SPIONs. Conclusion: This work shows how magnetic particle imaging can be enabling for new imaging and therapeutic applications of SPIONs in the lung.

A perspective on a rapid and radiation-free tracer imaging modality, magnetic particle imaging, with promise for clinical translation.

Magnetic particle imaging (MPI), introduced at the beginning of the twenty-first century, is emerging as a promising diagnostic tool in addition to the current repertoire of medical imaging modalities. Using superparamagnetic iron oxide nanoparticles (SPIOs), that are available for clinical use, MPI produces high contrast and highly sensitive tomographic images with absolute quantitation, no tissue attenuation at-depth, and there are no view limitations. The MPI signal is governed by the Brownian and Néel relaxation behavior of the particles. The relaxation time constants of these particles can be utilized to map information relating to the local microenvironment, such as viscosity and temperature. Proof-of-concept pre-clinical studies have shown favourable applications of MPI for better understanding the pathophysiology associated with vascular defects, tracking cell-based therapies and nanotheranostics. Functional imaging techniques using MPI will be useful for studying the pathology related to viscosity changes such as in vascular plaques and in determining cell viability of superparamagnetic iron oxide nanoparticle labeled cells. In this review article, an overview of MPI is provided with discussions mainly focusing on MPI tracers, applications of translational capabilities ranging from diagnostics to theranostics and finally outline a promising path towards clinical translation.

Magnetic particle imaging for radiation-free, sensitive and high-contrast vascular imaging and cell tracking.

Magnetic particle imaging (MPI) is an emerging ionizing radiation-free biomedical tracer imaging technique that directly images the intense magnetization of superparamagnetic iron oxide nanoparticles (SPIOs). MPI offers ideal image contrast because MPI shows zero signal from background tissues. Moreover, there is zero attenuation of the signal with depth in tissue, allowing for imaging deep inside the body quantitatively at any location. Recent work has demonstrated the potential of MPI for robust, sensitive vascular imaging and cell tracking with high contrast and dose-limited sensitivity comparable to nuclear medicine. To foster future applications in MPI, this new biomedical imaging field is welcoming researchers with expertise in imaging physics, magnetic nanoparticle synthesis and functionalization, nanoscale physics, and small animal imaging applications.

Magnetic Particle Imaging-Guided Heating in Vivo Using Gradient Fields for Arbitrary Localization of Magnetic Hyperthermia Therapy.

Image-guided treatment of cancer enables physicians to localize and treat tumors with great precision. Here, we present in vivo results showing that an emerging imaging modality, magnetic particle imaging (MPI), can be combined with magnetic hyperthermia into an image-guided theranostic platform. MPI is a noninvasive 3D tomographic imaging method with high sensitivity and contrast, zero ionizing radiation, and is linearly quantitative at any depth with no view limitations. The same superparamagnetic iron oxide nanoparticle (SPIONs) tracers imaged in MPI can also be excited to generate heat for magnetic hyperthermia. In this study, we demonstrate a theranostic platform, with quantitative MPI image guidance for treatment planning and use of the MPI gradients for spatial localization of magnetic hyperthermia to arbitrarily selected regions. This addresses a key challenge of conventional magnetic hyperthermia-SPIONs delivered systemically accumulate in off-target organs ( e.g., liver and spleen), and difficulty in localizing hyperthermia results in collateral heat damage to these organs. Using a MPI magnetic hyperthermia workflow, we demonstrate image-guided spatial localization of hyperthermia to the tumor while minimizing collateral damage to the nearby liver (1-2 cm distance). Localization of thermal damage and therapy was validated with luciferase activity and histological assessment. Apart from localizing thermal therapy, the technique presented here can also be extended to localize actuation of drug release and other biomechanical-based therapies. With high contrast and high sensitivity imaging combined with precise control and localization of the actuated therapy, MPI is a powerful platform for magnetic-based theranostics.

Magnetic Particle Imaging for Highly Sensitive, Quantitative, and Safe in Vivo Gut Bleed Detection in a Murine Model.

Gastrointestinal (GI) bleeding causes more than 300 000 hospitalizations per year in the United States. Imaging plays a crucial role in accurately locating the source of the bleed for timely intervention. Magnetic particle imaging (MPI) is an emerging clinically translatable imaging modality that images superparamagnetic iron-oxide (SPIO) tracers with extraordinary contrast and sensitivity. This linearly quantitative modality has zero background tissue signal and zero signal depth attenuation. MPI is also safe: there is zero ionizing radiation exposure to the patient and clinically approved tracers can be used with MPI. In this study, we demonstrate the use of MPI along with long-circulating, PEG-stabilized SPIOs for rapid in vivo detection and quantification of GI bleed. A mouse model genetically predisposed to GI polyp development (ApcMin/+) was used for this study, and heparin was used as an anticoagulant to induce acute GI bleeding. We then injected MPI-tailored, long-circulating SPIOs through the tail vein, and tracked the tracer biodistribution over time using our custom-built high resolution field-free line (FFL) MPI scanner. Dynamic MPI projection images captured tracer accumulation in the lower GI tract with excellent contrast. Quantitative analysis of the MPI images show that the mice experienced GI bleed rates between 1 and 5 µL/min. Although there are currently no human scale MPI systems, and MPI-tailored SPIOs need to undergo further development and evaluation, clinical translation of the technique is achievable. The robust contrast, sensitivity, safety, ability to image anywhere in the body, along with long-circulating SPIOs lends MPI outstanding promise as a clinical diagnostic tool for GI bleeding.

Seeing SPIOs Directly In Vivo with Magnetic Particle Imaging.

Magnetic particle imaging (MPI) is a new molecular imaging technique that directly images superparamagnetic tracers with high image contrast and sensitivity approaching nuclear medicine techniques-but without ionizing radiation. Since its inception, the MPI research field has quickly progressed in imaging theory, hardware, tracer design, and biomedical applications. Here, we describe the history and field of MPI, outline pressing challenges to MPI technology and clinical translation, highlight unique applications in MPI, and describe the role of the WMIS MPI Interest Group in collaboratively advancing MPI as a molecular imaging technique. We invite interested investigators to join the MPI Interest Group and contribute new insights and innovations to the MPI field.

Tracking short-term biodistribution and long-term clearance of SPIO tracers in magnetic particle imaging.

Magnetic particle imaging (MPI) is an emerging tracer-based medical imaging modality that images non-radioactive, kidney-safe superparamagnetic iron oxide (SPIO) tracers. MPI offers quantitative, high-contrast and high-SNR images, so MPI has exceptional promise for applications such as cell tracking, angiography, brain perfusion, cancer detection, traumatic brain injury and pulmonary imaging. In assessing MPI's utility for applications mentioned above, it is important to be able to assess tracer short-term biodistribution as well as long-term clearance from the body. Here, we describe the biodistribution and clearance for two commonly used tracers in MPI: Ferucarbotran (Meito Sangyo Co., Japan) and LS-oo8 (LodeSpin Labs, Seattle, WA). We successfully demonstrate that 3D MPI is able to quantitatively assess short-term biodistribution, as well as long-term tracking and clearance of these tracers in vivo.

Octreotide Functionalized Nano-Contrast Agent for Targeted Magnetic Resonance Imaging.

Reversible addition-fragmentation chain transfer (RAFT) polymerization has been employed to synthesize branched block copolymer nanoparticles possessing 1,4,7,10-tetraazacyclododecane-N,N,'N,″N,‴-tetraacetic acid (DO3A) macrocycles within their cores and octreotide (somatostatin mimic) cyclic peptides at their periphery. These polymeric nanoparticles have been chelated with Gd3+ and applied as magnetic resonance imaging (MRI) nanocontrast agents. This nanoparticle system has an r1 relaxivity of 8.3 mM-1 s-1, which is 3 times the r1 of commercial gadolinium-based contrast agents (GBCAs). The in vitro targeted binding efficiency of these nanoparticles shows 5 times greater affinity to somatostatin receptor type 2 (SSTR2) with Ki = 77 pM (compared to somatostatin with Ki = 0.385 nM). We have also evaluated the tumor targeting molecular imaging ability of these branched copolymer nanoparticle in vivo using nude/NCr mice bearing AR42J rat pancreatic tumor (SSTR2 positive) and A549 human lung carcinoma tumor (SSTR2 negative) xenografts.

Synthesis of Ferromagnetic Fe0.6 Mn0.4 O Nanoflowers as a New Class of Magnetic Theranostic Platform for In Vivo T1 -T2 Dual-Mode Magnetic Resonance Imaging and Magnetic Hyperthermia Therapy.

Uniform wüstite Fe0.6 Mn0.4 O nanoflowers have been successfully developed as an innovative theranostic agent with T1 -T2 dual-mode magnetic resonance imaging (MRI), for diagnostic applications and therapeutic interventions via magnetic hyperthermia. Unlike their antiferromagnetic bulk counterpart, the obtained Fe0.6 Mn0.4 O nanoflowers show unique room-temperature ferromagnetic behavior, probably due to the presence of an exchange coupling effect. Combined with the flower-like morphology, ferromagnetic Fe0.6 Mn0.4 O nanoflowers are demonstrated to possess dual-modal MRI sensitivity, with longitudinal relaxivity r1 and transverse relaxivity r2 as high as 4.9 and 61.2 mm(-1) s(-1) [Fe]+[Mn], respectively. Further in vivo MRI carried out on the mouse orthotopic glioma model revealed gliomas are clearly delineated in both T1 - and T2 -weighted MR images, after administration of the Fe0.6 Mn0.4 O nanoflowers. In addition, the Fe0.6 Mn0.4 O nanoflowers also exhibit excellent magnetic induction heating effects. Both in vitro and in vivo magnetic hyperthermia experimentation has demonstrated that magnetic hyperthermia by using the innovative Fe0.6 Mn0.4 O nanoflowers can induce MCF-7 breast cancer cell apoptosis and a complete tumor regression without appreciable side effects. The results have demonstrated that the innovative Fe0.6 Mn0.4 O nanoflowers can be a new magnetic theranostic platform for in vivo T1 -T2 dual-mode MRI and magnetic thermotherapy, thereby achieving a one-stop diagnosis cum effective therapeutic modality in cancer management.

Multispectral optoacoustic and MRI coregistration for molecular imaging of orthotopic model of human glioblastoma.

Multi-modality imaging methods are of great importance in oncologic studies for acquiring complementary information, enhancing the efficacy in tumor detection and characterization. We hereby demonstrate a hybrid non-invasive in vivo imaging approach of utilizing magnetic resonance imaging (MRI) and Multispectral Optoacoustic Tomography (MSOT) for molecular imaging of glucose uptake in an orthotopic glioblastoma in mouse. The molecular and functional information from MSOT can be overlaid on MRI anatomy via image coregistration to provide insights into probe uptake in the brain, which is verified by ex vivo fluorescence imaging and histological validation. In vivo MSOT and MRI imaging of an orthotopic glioma mouse model injected with IRDye800-2DG. Image coregistration between MSOT and MRI enables multifaceted (anatomical, functional, molecular) information from MSOT to be overlaid on MRI anatomy images to derive tumor physiological parameters such as perfusion, haemoglobin and oxygenation.