Inflammation has a key role in the pathogenesis of various human diseases. The early detection, localization and monitoring of inflammation are crucial for tailoring individual therapies. However, reliable biomarkers to detect local inflammatory activities and to predict disease outcome are still missing. Alarmins, which are locally released during cellular stress, are early amplifiers of inflammation. Here, using optical molecular imaging, we demonstrate that the alarmin S100A8/S100A9 serves as a sensitive local and systemic marker for the detection of even sub-clinical disease activity in inflammatory and immunological processes like irritative and allergic contact dermatitis. In a model of collagen-induced arthritis, we use S100A8/S100A9 imaging to predict the development of disease activity. Furthermore, S100A8/S100A9 can act as a very early and sensitive biomarker in experimental leishmaniasis for phagocyte activation linked to an effective Th1-response. In conclusion, the alarmin S100A8/S100A9 is a valuable and sensitive molecular target for novel imaging approaches to monitor clinically relevant inflammatory disorders on a molecular level.

• A handheld MSOT probe enables real-time molecular imaging of the breast. • MSOT of healthy controls provides a reproducible reference for pathology identification. • MSOT parameters allows for differentiation of invasive carcinoma and healthy tissue.

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This study aimed at evaluating hybrid multispectral optoacoustic tomography/ultrasound for imaging of thyroid disorders, including Graves' disease and thyroid nodules. Methods: The functional biomarkers and tissue parameters deoxygenated hemoglobin, oxygenated hemoglobin, total hemoglobin, saturation of hemoglobin, fat content, and water content were analyzed in thyroid lobes affected by Graves' disease (n 5 6), thyroid lobes with healthy tissue (n 5 8), benign thyroid nodules (n 5 13), and malignant thyroid nodules (n 5 3). Results: In Graves' disease, significantly higher deoxygenated hemoglobin (3.18 ± 0.52 vs. 2.13 ± 0.62; P 5 0.0055) and total hemoglobin (8.34 ± 0.88 vs. 6.59 ± 1.16; P 5 0.0084) and significantly lower fat content (0.64 ± 0.37 vs. 1.69 ± 1.25; P 5 0.0293) were found than in healthy controls. Malignant thyroid nodules showed significantly lower saturation of hemoglobin (55.4% ± 2.6% vs. 60.8% ± 7.2%; P 5 0.0393) and lower fat content (0.62 ± 0.19 vs. 1.46 ± 0.87; P 5 0.1295) than benign nodules. Conclusion: This pilot study showed the applicability and the potential of hybrid multispectral optoacoustic tomography/ultrasound to semiquantitatively provide tissue characterization and functional parameters in thyroid disorders for improved noninvasive diagnostics of thyroid diseases.

Near-infrared imaging such as fluorescence reflectance imaging (FRI) and fluorescence-mediated tomography (FMT) yields high signal-to-noise ratios (SNRs) and should thus be well suited for cell-tracking studies. Extravasation of monocytes or macrophages (Mfs) is one of the earliest events in inflammation. The purpose of this study was to assess whether FRI and FMT allow for the visualization and quantification of early inflammatory processes by tracing the migration of fluorescence-labeled murine Mfs in a cutaneous granuloma model. Methods: Mfs were labeled with a membrane-selective carbocyanine dye (1,3,3,). Cellular viability and function (nitric oxide production, phagocytosis, adherence) were assessed in vitro. Local inflammation was induced in mice by the subcutaneous injection of polyacrylamide gel pellets including or excluding a strong inflammatory stimulus (lipopolysaccharide). Labeled Mfs were injected intravenously, and FRI and FMT were performed up to 7 d. SNRs were calculated for the pellets, and the 3-dimensional distribution of Mfs was assessed using FMT. Cells were harvested from gel pellets and analyzed by flow cytometry. Results: DiR labeling did not affect cell viability or cell function. FRI revealed the migration of labeled Mfs into gel pellets and the homing of Mfs to different body compartments. The lipopolysaccharide-containing pellets exhibited significantly higher SNRs than did pellets without lipopolysaccharide. FMT showed that Mfs distributed mainly in the periphery of the pellets. Opt ical imaging is an appealing concept for studying cell migration in vivo. Specifically, in the near-infrared range (NIR) background fluorescence is minimal, yielding excellent signal-to-noise ratios (SNRs) and thus high sensitivity of the technique for detecting even small amounts of cells. Further, fluorophores emitting in the NIR can sufficiently penetrate the tissue, even from deeper sections, and allow detection over a longer time (1). Moreover, imaging techniques for optical imaging are rapidly developing, ranging from simple surface-weighted reflection (fluorescence reflectance imaging [FRI]) to fluorescence-mediated tomography (FMT) approaches. The latter technology is capable of delivering quantitative information on 3-dimensional fluorochrome distribution in vivo.The recruitment of immune cells and especially monocytes from the blood into tissue is crucial for the development and maintenance of inflammation (2). Interference with the mechanisms of monocyte extravasation represents an emerging new therapeutic strategy (3,4). After extravasation, monocytes can differentiate into macrophages and dendritic cells. They are crucial for nearly every step of an immune reaction including the initiation of inflammation, clearance of infectious agents or tumor cells, initiation of an adaptive immune response, and resolution of inflammation (5,6).Thus, the noninvasive tracing and monitoring of monocytes or macrophages (Mfs) in vivo by optical imaging could allow for the better localization, visualization, and

Time-lapse MRI was implemented for dynamic non-invasive cell tracking of individual slowly moving intravascular immune cells. Repetitive MRI acquisition enabled dynamic observation of iron oxide nanoparticle (ION) labelled cells. Simulations of MRI contrast indicated that only cells moving slower than 1 µm/s were detectable. Time-lapse MRI of the brain was performed after either IONs or ION-labelled monocytes were injected intravenously into naïve and experimental autoimmune encephalomyelitis (EAE) bearing mice at a presymptomatic or symptomatic stage. EAE mice showed a reduced number of slow moving, i.e. patrolling cells before and after onset of symptoms as compared to naïve controls. This observation is consistent with the notion of altered cell dynamics, i.e. higher velocities of immune cells rolling along the endothelium in the inflamed condition. Thus, time-lapse MRI enables for assessing immune cell dynamics non-invasively in deep tissue and may serve as a tool for detection or monitoring of an inflammatory response.

The study aimed to evaluate the clinical feasibility of hybrid ultrasound/multispectral optoacoustic tomography (MSOT) for assessing microvascular dysfunction in systemic sclerosis (SSc). A handheld US/MSOT imaging system was applied for imaging patients diagnosed with SSc (n = 7) and healthy volunteers (n = 8). Semiquantitative MSOT values for deoxygenated (HbR), oxygenated (HbO ) and total haemoglobin (HbT) were analysed for subcutaneous finger tissue of both hands (8 fingers per subject, 120 fingers in total) and used to assess disease activity (progressive vs stable). Grouped data were compared by one-way nested analysis of variance, Tukey post-hoc test as well as student's t test were used for statistical analysis.Subcutaneous finger tissue of patients with SSc provided significantly lower MSOT values for HbO (26.16 ± 0.71 vs 38.2 ± 1.54, P = .023) and HbT (55.92 ± 1.62 vs 72.46 ± 1.90, P = .018) compared to healthy volunteers. Patients with progressive SSc had significantly lower MSOT values compared to patients with stable disease and healthy volunteers.This pilot study shows the feasibility of MSOT imaging to resolve microvascular dysfunction in SSc as a marker of disease activity. By providing biological tissue properties not revealed by other imaging modalities, MSOT might help to grade SSc non-invasively and monitor early therapy response.

IMPORTANCE Differential diagnosis of congenital vascular anomalies is challenging, and misdiagnosis is frequent. Vascular malformations are considered one of the most difficult vascular diseases to treat. A new imaging approach that visualizes anatomical features and quantitatively assesses molecular biomarkers noninvasively would aid diagnosis and monitoring of treatment response of vascular malformations.OBJECTIVE To evaluate multispectral optoacoustic tomography (MSOT) for noninvasive assessment of molecular biomarkers for diagnosis and therapeutic monitoring of vascular malformations. DESIGN, SETTING, AND PARTICIPANTSThis pilot study examined 6 patients with arteriovenous malformation (AVM) and 6 patients with venous malformation (VM) diagnosed according to the classification system of the International Society for the Study of Vascular Anomalies. All patients underwent clinical hybrid MSOT/ultrasonographic (US) imaging before and after treatment at an interdisciplinary vascular malformations clinic by trained MSOT and US examiners. Examiners were blinded to the patient history and stage of disease.