Institute of Biological and Medical Imaging

Originally developed for diagnostic ultrasound imaging, piezoelectric transducers are the most widespread technology employed in optoacoustic (photoacoustic) signal detection. However, the detection requirements of optoacoustic sensing and imaging differ from those of conventional ultrasonography and lead to specifications not sufficiently addressed by piezoelectric detectors. Consequently, interest has shifted to utilizing entirely optical methods for measuring optoacoustic waves. All-optical sound detectors yield a higher signal-to-noise ratio per unit area than piezoelectric detectors and feature wide detection bandwidths that may be more appropriate for optoacoustic applications, enabling several biomedical or industrial applications. Additionally, optical sensing of sound is less sensitive to electromagnetic noise, making it appropriate for a greater spectrum of environments. In this review, we categorize different methods of optical ultrasound detection and discuss key technology trends geared towards the development of all-optical optoacoustic systems. We also review application areas that are enabled by all-optical sound detectors, including interventional imaging, non-contact measurements, magnetoacoustics, and non-destructive testing.

BACKGROUND: Early repolarization pattern (ERP) on electrocardiogram was associated with idiopathic ventricular fibrillation and sudden cardiac arrest in a case-control study and with cardiovascular mortality in a Finnish community-based sample. We sought to determine ERP prevalence and its association with cardiac and all-cause mortality in a large, prospective, population-based case-cohort study (Monitoring of Cardiovascular Diseases and Conditions [MONICA]/KORA [Cooperative Health Research in the Region of Augsburg]) comprised of individuals of Central-European descent. METHODS AND FINDINGS: Electrocardiograms of 1,945 participants aged 35-74 y, representing a source population of 6,213 individuals, were analyzed applying a case-cohort design. Mean follow-up was 18.9 y. Cause of death was ascertained by the 9th revision of the International Classification of Disease (ICD-9) codes as documented in death certificates. ERP-attributable effects on mortality were determined by a weighted Cox proportional hazard model adjusted for covariables. Prevalence of ERP was 13.1% in our study. ERP was associated with cardiac and all-cause mortality, most pronounced in those of younger age and male sex; a clear ERP-age interaction was detected (p = 0.005). Age-stratified analyses showed hazard ratios (HRs) for cardiac mortality of 1.96 (95% confidence interval [CI] 1.05-3.68, p = 0.035) for both sexes and 2.65 (95% CI 1.21-5.83, p = 0.015) for men between 35-54 y. An inferior localization of ERP further increased ERP-attributable cardiac mortality to HRs of 3.15 (95% CI 1.58-6.28, p = 0.001) for both sexes and to 4.27 (95% CI 1.90-9.61, p<0.001) for men between 35-54 y. HRs for all-cause mortality were weaker but reached significance. CONCLUSIONS: We found a high prevalence of ERP in our population-based cohort of middle-aged individuals. ERP was associated with about a 2- to 4-fold increased risk of cardiac mortality in individuals between 35 and 54 y. An inferior localization of ERP was associated with a particularly increased risk. Please see later in the article for the Editors' Summary.

Light propagating in tissue attains a spectrum that varies with location due to wavelength-dependent fluence attenuation, an effect that causes spectral corruption. Spectral corruption has limited the quantification accuracy of optical and optoacoustic spectroscopic methods, and impeded the goal of imaging blood oxygen saturation (sO2) deep in tissues; a critical goal for the assessment of oxygenation in physiological processes and disease. Here we describe light fluence in the spectral domain and introduce eigenspectra multispectral optoacoustic tomography (eMSOT) to account for wavelength-dependent light attenuation, and estimate blood sO2 within deep tissue. We validate eMSOT in simulations, phantoms and animal measurements and spatially resolve sO2 in muscle and tumours, validating our measurements with histology data. eMSOT shows substantial sO2 accuracy enhancement over previous optoacoustic methods, potentially serving as a valuable tool for imaging tissue pathophysiology.

Abstract Advances in genetic engineering have enabled the use of bacterial outer membrane vesicles (OMVs) to deliver vaccines, drugs and immunotherapy agents, as a strategy to circumvent biocompatibility and large-scale production issues associated with synthetic nanomaterials. We investigate bioengineered OMVs for contrast enhancement in optoacoustic (photoacoustic) imaging. We produce OMVs encapsulating biopolymer-melanin (OMV Mel ) using a bacterial strain expressing a tyrosinase transgene. Our results show that upon near-infrared light irradiation, OMV Mel generates strong optoacoustic signals appropriate for imaging applications. In addition, we show that OMV Mel builds up intense heat from the absorbed laser energy and mediates photothermal effects both in vitro and in vivo. Using multispectral optoacoustic tomography, we noninvasively monitor the spatio-temporal, tumour-associated OMV Mel distribution in vivo. This work points to the use of bioengineered vesicles as potent alternatives to synthetic particles more commonly employed for optoacoustic imaging, with the potential to enable both image enhancement and photothermal applications.

Higher resting heart rate is associated with increased cardiovascular disease and mortality risk. Though heritable factors play a substantial role in population variation, little is known about specific genetic determinants. This knowledge can impact clinical care by identifying novel factors that influence pathologic heart rate states, modulate heart rate through cardiac structure and function or by improving our understanding of the physiology of heart rate regulation. To identify common genetic variants associated with heart rate, we performed a meta-analysis of 15 genome-wide association studies (GWAS), including 38,991 subjects of European ancestry, estimating the association between age-, sex- and body mass-adjusted RR interval (inverse heart rate) and approximately 2.5 million markers. Results with P < 5 × 10(-8) were considered genome-wide significant. We constructed regression models with multiple markers to assess whether results at less stringent thresholds were likely to be truly associated with RR interval. We identified six novel associations with resting heart rate at six loci: 6q22 near GJA1; 14q12 near MYH7; 12p12 near SOX5, c12orf67, BCAT1, LRMP and CASC1; 6q22 near SLC35F1, PLN and c6orf204; 7q22 near SLC12A9 and UfSp1; and 11q12 near FADS1. Associations at 6q22 400 kb away from GJA1, at 14q12 MYH6 and at 1q32 near CD34 identified in previously published GWAS were confirmed. In aggregate, these variants explain approximately 0.7% of RR interval variance. A multivariant regression model including 20 variants with P < 10(-5) increased the explained variance to 1.6%, suggesting that some loci falling short of genome-wide significance are likely truly associated. Future research is warranted to elucidate underlying mechanisms that may impact clinical care.

We genetically controlled compartmentalization in eukaryotic cells by heterologous expression of bacterial encapsulin shell and cargo proteins to engineer enclosed enzymatic reactions and size-constrained metal biomineralization. The shell protein (EncA) from Myxococcus xanthus auto-assembles into nanocompartments inside mammalian cells to which sets of native (EncB,C,D) and engineered cargo proteins self-target enabling localized bimolecular fluorescence and enzyme complementation. Encapsulation of the enzyme tyrosinase leads to the confinement of toxic melanin production for robust detection via multispectral optoacoustic tomography (MSOT). Co-expression of ferritin-like native cargo (EncB,C) results in efficient iron sequestration producing substantial contrast by magnetic resonance imaging (MRI) and allowing for magnetic cell sorting. The monodisperse, spherical, and iron-loading nanoshells are also excellent genetically encoded reporters for electron microscopy (EM). In general, eukaryotically expressed encapsulins enable cellular engineering of spatially confined multicomponent processes with versatile applications in multiscale molecular imaging, as well as intriguing implications for metabolic engineering and cellular therapy.

Imaging has become an indispensable tool in the research and clinical management of cardiovascular disease (CVD). An array of imaging technologies is considered for CVD diagnostics and therapeutic assessment, ranging from ultrasonography, X-ray computed tomography and magnetic resonance imaging to nuclear and optical imaging methods. Each method has different operational characteristics and assesses different aspects of CVD pathophysiology; nevertheless, more information is desirable for achieving a comprehensive view of the disease. Optoacoustic (photoacoustic) imaging is an emerging modality promising to offer novel information on CVD parameters by allowing high-resolution imaging of optical contrast several centimeters deep inside tissue. Implemented with illumination at several wavelengths, multi-spectral optoacoustic tomography (MSOT) in particular, is sensitive to oxygenated and deoxygenated hemoglobin, water and lipids allowing imaging of the vasculature, tissue oxygen saturation and metabolic or inflammatory parameters. Progress with fast-tuning lasers, parallel detection and advanced image reconstruction and data-processing algorithms have recently transformed optoacoustics from a laboratory tool to a promising modality for small animal and clinical imaging. We review progress with optoacoustic CVD imaging, highlight the research and diagnostic potential and current applications and discuss the advantages, limitations and possibilities for integration into clinical routine.

Adipocyte-derived extracellular vesicles (AdEVs) are membranous nanoparticles that convey communication from adipose tissue to other organs. Here, to delineate their role as messengers with glucoregulatory nature, we paired fluorescence AdEV-tracing and SILAC-labeling with (phospho)proteomics, and revealed that AdEVs transfer functional insulinotropic protein cargo into pancreatic β-cells. Upon transfer, AdEV proteins were subjects for phosphorylation, augmented insulinotropic GPCR/cAMP/PKA signaling by increasing total protein abundances and phosphosite dynamics, and ultimately enhanced 1st-phase glucose-stimulated insulin secretion (GSIS) in murine islets. Notably, insulinotropic effects were restricted to AdEVs isolated from obese and insulin resistant, but not lean mice, which was consistent with differential protein loads and AdEV luminal morphologies. Likewise, in vivo pre-treatment with AdEVs from obese but not lean mice amplified insulin secretion and glucose tolerance in mice. This data suggests that secreted AdEVs can inform pancreatic β-cells about insulin resistance in adipose tissue in order to amplify GSIS in times of increased insulin demand.

We report on a new quantification methodology of optoacoustic tomographic reconstructions under heterogeneous illumination conditions representative of realistic whole-body imaging scenarios. Our method relies on the differences in the spatial characteristics of the absorption coefficient and the optical energy density within the medium. By using sparse-representation based decomposition, we exploit these different characteristics to extract both the absorption coefficient and the photon density within the imaged object from the optoacoustic image. In contrast to previous methods, this algorithm is not based on the solution of theoretical light transport equations and it does not require explicit knowledge of the illumination geometry or the optical properties of the object and other unknown or loosely defined experimental parameters, leading to highly robust performance. The method was successfully examined with numerically and experimentally generated data and was found to be ideally suited for practical implementations in tomographic schemes of varying complexity, including multiprojection illumination systems and multispectral optoacoustic tomography (MSOT) studies of tissue biomarkers.

Due to loss of tactile feedback the assessment of tumor margins during robotic surgery is based only on visual inspection, which is neither significantly sensitive nor specific. Here we demonstrate time-resolved fluorescence spectroscopy (TRFS) as a novel technique to complement the visual inspection of oral cancers during transoral robotic surgery (TORS) in real-time and without the need for exogenous contrast agents. TRFS enables identification of cancerous tissue by its distinct autofluorescence signature that is associated with the alteration of tissue structure and biochemical profile. A prototype TRFS instrument was integrated synergistically with the da Vinci Surgical robot and the combined system was validated in swine and human patients. Label-free and real-time assessment and visualization of tissue biochemical features during robotic surgery procedure, as demonstrated here, not only has the potential to improve the intraoperative decision making during TORS but also other robotic procedures without modification of conventional clinical protocols.

Existing mammographic screening solutions are generally associated with several major drawbacks, such as exposure to ionizing radiation or insufficient sensitivity in younger populations with radiographically-dense breast. Even when combined with ultrasound or magnetic resonance imaging, X-Ray mammography may still attain unspecific or false positive results. Thus, development of new breast imaging tools represents a timely medical challenge. We report on a new approach to high-resolution functional and anatomical breast angiography using volumetric hand-held optoacoustic tomography, which employs light intensities safe for human use. Experiments in young healthy volunteers with fibroglandular-dominated dense breasts revealed the feasibility of rendering three-dimensional images representing vascular anatomy and functional blood oxygenation parameters at video rate. Sufficient contrast was achieved at depths beyond 2 cm within dense breasts without compromising the real-time imaging performance. The suggested solution may thus find applicability as a standalone or supplemental screening tool for early detection and follow-up of carcinomas in radiographically-dense breasts. Volumetric handheld optoacoustic tomography scanner uses safe pulses of near-infrared light to render three-dimensional images of deep vascular anatomy, blood oxygenation and breast parenchyma at video rate.

This study aimed at evaluating hybrid multispectral optoacoustic tomography/ultrasound for imaging of thyroid disorders, including Graves’ disease and thyroid nodules. <b>Methods:</b> 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 (<i>n</i> = 6), thyroid lobes with healthy tissue (<i>n</i> = 8), benign thyroid nodules (<i>n</i> = 13), and malignant thyroid nodules (<i>n</i> = 3). <b>Results:</b> In Graves’ disease, significantly higher deoxygenated hemoglobin (3.18 ± 0.52 vs. 2.13 ± 0.62; <i>P</i> = 0.0055) and total hemoglobin (8.34 ± 0.88 vs. 6.59 ± 1.16; <i>P</i> = 0.0084) and significantly lower fat content (0.64 ± 0.37 vs. 1.69 ± 1.25; <i>P</i> = 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%; <i>P</i> = 0.0393) and lower fat content (0.62 ± 0.19 vs. 1.46 ± 0.87; <i>P</i> = 0.1295) than benign nodules. <b>Conclusion:</b> 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.

Non-invasive glucose monitoring (NIGM) represents an attractive alternative to finger pricking for blood glucose assessment and management of diabetes. Nevertheless, current NIGM techniques do not measure glucose concentrations in blood but rely on indirect bulk measurement of glucose in interstitial fluid, where glucose is diluted and glucose dynamics are different from those in the blood, which impairs NIGM accuracy. Here we introduce a new biosensor, termed depth-gated mid-infrared optoacoustic sensor (DIROS), which allows, for the first time, non-invasive glucose detection in blood-rich volumes in the skin. DIROS minimizes interference caused by the stratum corneum and other superficial skin layers by time-gating mid-infrared optoacoustic signals to enable depth-selective localization of glucose readings in skin. In measurements on the ears of (female) mice, DIROS displays improved accuracy over bulk-tissue glucose measurements. Our work demonstrates how signal localization can improve NIGM accuracy and positions DIROS as a holistic approach, with high translational potential, that addresses a key limitation of current NIGM methods.

The characteristics of tumour development and metastasis relate not only to genomic heterogeneity but also to spatial heterogeneity, associated with variations in the intratumoural arrangement of cell populations, vascular morphology and oxygen and nutrient supply. While optical (photonic) microscopy is commonly employed to visualize the tumour microenvironment, it assesses only a few hundred cubic microns of tissue. Therefore, it is not suitable for investigating biological processes at the level of the entire tumour, which can be at least four orders of magnitude larger. In this study, we aimed to extend optical visualization and resolve spatial heterogeneity throughout the entire tumour volume. We developed an optoacoustic (photoacoustic) mesoscope adapted to solid tumour imaging and, in a pilot study, offer the first insights into cancer optical contrast heterogeneity in vivo at an unprecedented resolution of <50 μm throughout the entire tumour mass. Using spectral methods, we resolve unknown patterns of oxygenation, vasculature and perfusion in three types of breast cancer and showcase different levels of structural and functional organization. To our knowledge, these results are the most detailed insights of optical signatures reported throughout entire tumours in vivo, and they position optoacoustic mesoscopy as a unique investigational tool linking microscopic and macroscopic observations.

A method is presented to reduce artefacts produced in optoacoustic tomography images due to internal reflection or scattering of the acoustic waves. It is based on weighting the tomographic contribution of each detector with the probability that a signal affected by acoustic mismatches is measured at that position. The correction method does not require a priori knowledge of the acoustic or optical properties of the imaged sample. Performance tests were made with agar phantoms that included air gaps for mimicking strong acoustic reflections as well as with an acoustically heterogeneous adult Zebrafish. The results obtained with the method proposed show a clear reduction of the artefacts with respect to the original images reconstructed with filtered back-projection algorithm. This performance is directly related to in vivo small animal imaging applications involving imaging in the presence of bones, lungs, and other highly mismatched organs.

Fluorescence imaging is currently being actively developed for surgical guidance; however, it remains underutilized for diagnostic and endoscopic surveillance of incipient colorectal cancer in high-risk patients. Here we demonstrate the utility and potential for clinical translation of a fluorescently labeled cathepsin-activated chemical probe to highlight gastrointestinal lesions. This probe stays optically dark until it is activated by proteases produced by tumor-associated macrophages and accumulates within the lesions, enabling their detection using an endoscope outfitted with a fluorescence detector. We evaluated the probe in multiple murine models and a human-scale porcine model of gastrointestinal carcinogenesis. The probe provides fluorescence-guided surveillance of gastrointestinal lesions and augments histopathological analysis by highlighting areas of dysplasia as small as 400 µm, which were visibly discernible with significant tumor-to-background ratios, even in tissues with a background of severe inflammation and ulceration. Given these results, we anticipate that this probe will enable sensitive fluorescence-guided biopsies, even in the presence of highly inflamed colorectal tissue, which will improve early diagnosis to prevent gastrointestinal cancers.

Abstract Optical microscopy remains a fundamental tool for modern biological discovery owing to its excellent spatial resolution and versatile contrast in visualizing cellular and sub-cellular structures. Yet, the time domain is paramount for the observation of biological dynamics in living systems. Commonly, acquisition of microscopy data involves scanning of a spherically- or cylindrically-focused light beam across the imaged volume, which significantly limits temporal resolution in 3D. Additional complications arise from intense light scattering of biological tissues, further restraining the effective penetration depth and field of view of optical microscopy techniques. To overcome these limitations, we devised a fast optoacoustic micro-tomography (OMT) approach based on simultaneous acquisition of 3D image data with a high-density hemispherical ultrasound array having effective detection bandwidth beyond 25 MHz. We demonstrate fast three-dimensional imaging of freely-swimming zebrafish larvae, achieving 3D imaging speed of 100 volumes per second with isotropic spatial resolution approaching the dimensions of large cells across a field of view exceeding 50mm 3 . As opposed to other microscopy techniques based on optical contrast, OMT resolves optical absorption acoustically using unfocused light excitation. Thus, no penetration barriers are imposed by light scattering in deep tissues, suggesting it as a powerful approach for multi-scale functional and molecular imaging applications.

Abstract Near‐infrared (NIR) light absorbing theranostic agents can integrate optoacoustic imaging and photothermal therapy for effective personalized precision medicine. However, most of these agents face the challenges of unstable optical properties, material‐associated toxicity, and nonbiodegradability, all of which limit their biomedical application. Several croconaine‐based organic agents able to overcome some of these limitations have been recently reported, but these suffer from complicated multistep synthesis protocols. Herein, the use of CR760, a croconaine dye with excellent optical properties, is reported for nanoparticle formulation and subsequent optoacoustic imaging and photothermal therapy. Importantly, CR760 can be conveniently prepared in a single step from commercially available materials. Furthermore, CR760 can be covalently attached, via a polyethylene glycol linker, to the α v β 3 integrin ligand c(RGDyC), resulting in self‐assembled nanoparticles (NPs) with cancer‐targeting capability. Such CR760RGD‐NPs exhibit strong NIR absorption, high photostability, high optoacoustic generation efficiency, and active tumor‐targeting, making them ideal candidates for optoacoustic imaging. Due to favorable electron transfer, CR760RGD‐NPs display a 45.37% photothermal conversion efficiency thereby rendering them additionally useful for photothermal therapy. Targeted tumor elimination, biosafety, and biocompatibility are demonstrated in a 4T1 murine breast tumor model. This work points to the use of CR760RGD‐NPs as a promising nanoagent for NIR‐based cancer phototheranostics.

Abstract Reversibly photo-switchable proteins are essential for many super-resolution fluorescence microscopic and optoacoustic imaging methods. However, they have yet to be used as sensors that measure the distribution of specific analytes at the nanoscale or in the tissues of live animals. Here we constructed the prototype of a photo-switchable Ca 2+ sensor based on GCaMP5G that can be switched with 405/488-nm light and describe its molecular mechanisms at the structural level, including the importance of the interaction of the core barrel structure of the fluorescent protein with the Ca 2+ receptor moiety. We demonstrate super-resolution imaging of Ca 2+ concentration in cultured cells and optoacoustic Ca 2+ imaging in implanted tumor cells in mice under controlled Ca 2+ conditions. Finally, we show the generalizability of the concept by constructing examples of photo-switching maltose and dopamine sensors based on periplasmatic binding protein and G-protein-coupled receptor-based sensors.

Several imaging techniques aim at identifying features of carotid plaque instability but come with limitations, such as the use of contrast agents, long examination times and poor portability. Multispectral optoacoustic tomography (MSOT) employs light and sound to resolve lipid and hemoglobin content, both features associated with plaque instability, in a label-free, fast and highly portable way. Herein, 5 patients with carotid atherosclerosis, 5 healthy volunteers and 2 excised plaques, were scanned with handheld MSOT. Spectral unmixing allowed visualization of lipid and hemoglobin content within three ROIs: whole arterial cross-section, plaque and arterial lumen. Calculation of the fat-blood-ratio (FBR) value within the ROIs enabled the differentiation between patients and healthy volunteers (P = 0.001) and between plaque and lumen in patients (P = 0.04). Our results introduce MSOT as a tool for molecular imaging of human carotid atherosclerosis and open new possibilities for research and clinical assessment of carotid plaques.