Skeletal muscle optoacoustics reveals patterns of circulatory function and oxygen metabolism during exercise.

Imaging skeletal muscle function and metabolism, as reported by local hemodynamics and oxygen kinetics, can elucidate muscle performance, severity of an underlying disease or outcome of a treatment. Herein, we used multispectral optoacoustic tomography (MSOT) to image hemodynamics and oxygen kinetics within muscle during exercise. Four healthy volunteers underwent three different hand-grip exercise challenges (60s isometric, 120s intermittent isometric and 60s isotonic). During isometric contraction, MSOT showed a decrease of HbO2, Hb and total blood volume (TBV), followed by a prominent increase after the end of contraction. Corresponding hemodynamic behaviors were recorded during the intermittent isometric and isotonic exercises. A more detailed analysis of MSOT readouts revealed insights into arteriovenous oxygen differences and muscle oxygen consumption during all exercise schemes. These results demonstrate an excellent capability of visualizing both circulatory function and oxygen metabolism within skeletal muscle under exercise, with great potential implications for muscle research, including relevant disease diagnostics.

Non-invasive multispectral optoacoustic tomography resolves intrahepatic lipids in patients with hepatic steatosis.

Hepatic steatosis is characterized by intrahepatic lipid accumulation and may lead to irreversible liver damage if untreated. Here, we investigate whether multispectral optoacoustic tomography (MSOT) can offer label-free detection of liver lipid content to enable non-invasive characterization of hepatic steatosis by analyzing the spectral region around 930 nm, where lipids characteristically absorb. In a pilot study, we apply MSOT to measure liver and surrounding tissues in five patients with liver steatosis and five healthy volunteers, revealing significantly higher absorptions at 930 nm in the patients, while no significant difference was observed in the subcutaneous adipose tissue of the two groups. We further corroborated the human observations with corresponding MSOT measurements in high fat diet (HFD) - and regular chow diet (CD)-fed mice. This study introduces MSOT as a potential non-invasive and portable technique for detecting/monitoring hepatic steatosis in clinical settings, providing justification for larger studies.

Mass spectrometry imaging discriminates glioblastoma tumor cell subpopulations and different microvascular formations based on their lipid profiles.

Glioblastoma is a prevalent malignant brain tumor and despite clinical intervention, tumor recurrence is frequent and usually fatal. Genomic investigations have provided a greater understanding of molecular heterogeneity in glioblastoma, yet there are still no curative treatments, and the prognosis has remained unchanged. The aggressive nature of glioblastoma is attributed to the heterogeneity in tumor cell subpopulations and aberrant microvascular proliferation. Ganglioside-directed immunotherapy and membrane lipid therapy have shown efficacy in the treatment of glioblastoma. To truly harness these novel therapeutics and develop a regimen that improves clinical outcome, a greater understanding of the altered lipidomic profiles within the glioblastoma tumor microenvironment is urgently needed. In this work, high resolution mass spectrometry imaging was utilized to investigate lipid heterogeneity in human glioblastoma samples. Data presented offers the first insight into the histology-specific accumulation of lipids involved in cell metabolism and signaling. Cardiolipins, phosphatidylinositol, ceramide-1-phosphate, and gangliosides, including the glioblastoma stem cell marker, GD3, were shown to differentially accumulate in tumor and endothelial cell subpopulations. Conversely, a reduction in sphingomyelins and sulfatides were detected in tumor cell regions. Cellular accumulation for each lipid class was dependent upon their fatty acid residue composition, highlighting the importance of understanding lipid structure-function relationships. Discriminating ions were identified and correlated to histopathology and Ki67 proliferation index. These results identified multiple lipids within the glioblastoma microenvironment that warrant further investigation for the development of predictive biomarkers and lipid-based therapeutics.

Weighted model-based optoacoustic reconstruction for partial-view geometries.

Acoustic heterogeneities in biological samples are known to cause artifacts in tomographic optoacoustic (photoacoustic) image reconstruction. A statistical weighted model-based reconstruction approach was previously introduced to mitigate such artifacts. However, this approach does not reliably provide high-quality reconstructions for partial-view imaging systems, which are common in preclinical and clinical optoacoustics. In this article, the capability of the weighted model-based algorithm is extended to generate optoacoustic reconstructions with less distortions for partial-view geometry data. This is achieved by manipulating the weighting scheme based on the detector geometry. Using partial-view optoacoustic tomography data from a tissue-mimicking phantom containing a strong acoustic reflector, tumors grafted onto mice, and a mouse brain with intact skull, the proposed partial-view-corrected weighted model-based algorithm is shown to mitigate reflection artifacts in reconstructed images without distorting structures or boundaries, compared with both conventional model-based and the weighted model-based algorithms. It is also demonstrated that the partial-view-corrected weighted model-based algorithm has the additional advantage of suppressing streaking artifacts due to the partial-view geometry itself in the presence of a very strong optoacoustic chromophore. Due to its enhanced performance, the partial-view-corrected weighted model-based algorithm may prove useful for improving the quality of partial-view multispectral optoacoustic tomography, leading to enhanced visualization of functional parameters such as tissue oxygenation.

Multi-Aspect Optoacoustic Imaging of Breast Tumors under Chemotherapy with Exogenous and Endogenous Contrasts: Focus on Apoptosis and Hypoxia.

Breast cancer is a complex tumor type involving many biological processes. Most chemotherapeutic agents exert their antitumoral effects by rapid induction of apoptosis. Another main feature of breast cancer is hypoxia, which may drive malignant progression and confer resistance to various forms of therapy. Thus, multi-aspect imaging of both tumor apoptosis and oxygenation in vivo would be of enormous value for the effective evaluation of therapy response. Herein, we demonstrate the capability of a hybrid imaging modality known as multispectral optoacoustic tomography (MSOT) to provide high-resolution, simultaneous imaging of tumor apoptosis and oxygenation, based on both the exogenous contrast of an apoptosis-targeting dye and the endogenous contrast of hemoglobin. MSOT imaging was applied on mice bearing orthotopic 4T1 breast tumors before and following treatment with doxorubicin. Apoptosis was monitored over time by imaging the distribution of xPLORE-APOFL750©, a highly sensitive poly-caspase binding apoptotic probe, within the tumors. Oxygenation was monitored by tracking the distribution of oxy- and deoxygenated hemoglobin within the same tumor areas. Doxorubicin treatment induced an increase in apoptosis-depending optoacoustic signal of xPLORE-APOFL750© at 24 h after treatment. Furthermore, our results showed spatial correspondence between xPLORE-APO750© and deoxygenated hemoglobin. In vivo apoptotic status of the tumor tissue was independently verified by ex vivo fluorescence analysis. Overall, our results provide a rationale for the use of MSOT as an effective tool for simultaneously investigating various aspects of tumor pathophysiology and potential effects of therapeutic regimes based on both endogenous and exogenous molecular contrasts.

Chemotherapeutic effects on breast tumor hemodynamics revealed by eigenspectra multispectral optoacoustic tomography (eMSOT).

Non-invasive monitoring of hemodynamic tumor responses to chemotherapy could provide unique insights into the development of therapeutic resistance and inform therapeutic decision-making in the clinic. Methods: Here, we examined the longitudinal and dynamic effects of the common chemotherapeutic drug Taxotere on breast tumor (KPL-4) blood volume and oxygen saturation using eigenspectra multispectral optoacoustic tomography (eMSOT) imaging over a period of 41 days. Tumor vascular function was assessed by dynamic oxygen-enhanced eMSOT (OE-eMSOT). The obtained in vivo optoacoustic data were thoroughly validated by ex vivo cryoimaging and immunohistochemical staining against markers of vascularity and hypoxia. Results: We provide the first preclinical evidence that prolonged treatment with Taxotere causes a significant drop in mean whole tumor oxygenation. Furthermore, application of OE-eMSOT showed a diminished vascular response in Taxotere-treated tumors and revealed the presence of static blood pools, indicating increased vascular permeability. Conclusion: Our work has important translational implications and supports the feasibility of eMSOT imaging for non-invasive assessment of tumor microenvironmental responses to chemotherapy.

Multispectral optoacoustic tomography of lipid and hemoglobin contrast in human carotid atherosclerosis.

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.

Resolution of Spatial and Temporal Heterogeneity in Bevacizumab-Treated Breast Tumors by Eigenspectra Multispectral Optoacoustic Tomography.

Understanding temporal and spatial hemodynamic heterogeneity as a function of tumor growth or therapy affects the development of novel therapeutic strategies. In this study, we employed eigenspectra multispectral optoacoustic tomography (eMSOT) as a next-generation optoacoustic method to impart high accuracy in resolving tumor hemodynamics during bevacizumab therapy in two types of breast cancer xenografts (KPL-4 and MDA-MB-468). Patterns of tumor total hemoglobin concentration (THb) and oxygen saturation (sO2) were imaged in control and bevacizumab-treated tumors over the course of 58 days (KPL-4) and 16 days (MDA-MB-468), and the evolution of functional vasculature "normalization" was resolved macroscopically. An initial sharp drop in tumor sO2 and THb content shortly after the initiation of bevacizumab treatment was followed by a recovery in oxygenation levels. Rim-core subregion analysis revealed steep spatial oxygenation gradients in growing tumors that were reduced after bevacizumab treatment. Critically, eMSOT imaging findings were validated directly by histopathologic assessment of hypoxia (pimonidazole) and vascularity (CD31). These data demonstrate how eMSOT brings new abilities for accurate observation of entire tumor responses to challenges at spatial and temporal dimensions not available by other techniques today. SIGNIFICANCE: Accurate assessment of hypoxia and vascularization over space and time is critical for understanding tumor development and the role of spatial heterogeneity in tumor aggressiveness, metastasis, and response to treatment.

Multispectral optoacoustic tomography of muscle perfusion and oxygenation under arterial and venous occlusion: A human pilot study.

Perfusion and oxygenation are critical parameters of muscle metabolism in health and disease. They have been both the target of many studies, in particular using near-infrared spectroscopy (NIRS). However, difficulties with quantifying NIRS signals have limited a wide dissemination of the method to the clinics. Our aim was to investigate whether clinical multispectral optoacoustic tomography (MSOT) could enable the label-free imaging of muscle perfusion and oxygenation under clinically relevant challenges: the arterial and venous occlusion. We employed a hybrid clinical MSOT/ultrasound system equipped with a hand-held scanning probe to visualize hemodynamic and oxygenation changes in skeletal muscle under arterial and venous occlusions. Four (N = 4) healthy volunteers were scanned over the forearm for both 3-minute occlusion challenges. MSOT-recorded pathophysiologically expected results during tests of disturbed blood flow with high resolution and without the need for contrast agents. During arterial occlusion, MSOT-extracted Hb-values showed an increase, while HbO2 - and total blood volume (TBV)-values remained roughly steady, followed by a discrete increase during the hyperemic period after cuff deflation. During venous occlusion, results showed a clear increase in intramuscular HbO2 , Hb and TBV within the segmented muscle area. MSOT was found to be capable of label-free non-invasive imaging of muscle hemodynamics and oxygenation under arterial and venous occlusion. We introduce herein MSOT as a novel modality for the assessment of vascular disorders characterized by disturbed blood flow, such as acute limb ischemia and venous thrombosis.

Fluorescence imaging reversion using spatially variant deconvolution.

Fluorescence imaging opens new possibilities for intraoperative guidance and early cancer detection, in particular when using agents that target specific disease features. Nevertheless, photon scattering in tissue degrades image quality and leads to ambiguity in fluorescence image interpretation and challenges clinical translation. We introduce the concept of capturing the spatially-dependent impulse response of an image and investigate Spatially Adaptive Impulse Response Correction (SAIRC), a method that is proposed for improving the accuracy and sensitivity achieved. Unlike classical methods that presume a homogeneous spatial distribution of optical properties in tissue, SAIRC explicitly measures the optical heterogeneity in tissues. This information allows, for the first time, the application of spatially-dependent deconvolution to correct the fluorescence images captured in relation to their modification by photon scatter. Using experimental measurements from phantoms and animals, we investigate the improvement in resolution and quantification over non-corrected images. We discuss how the proposed method is essential for maximizing the performance of fluorescence molecular imaging in the clinic.

Multispectral Optoacoustic Tomography of Brown Adipose Tissue.

MSOT has revolutionized biomedical imaging because it allows anatomical, functional, and molecular imaging of deep tissues in vivo in an entirely noninvasive, label-free, and real-time manner. This imaging modality works by pulsing light onto tissue, triggering the production of acoustic waves, which can be collected and reconstructed to provide high-resolution images of features as deep as several centimeters below the body surface. Advances in hardware and software continue to bring MSOT closer to clinical translation. Most recently, a clinical handheld MSOT system has been used to image brown fat tissue (BAT) and its metabolic activity by directly resolving the spectral signatures of hemoglobin and lipids. This opens up new possibilities for studying BAT physiology and its role in metabolic disease without the need to inject animals or humans with contrast agents. In this chapter, we overview how MSOT works and how it has been implemented in preclinical and clinical contexts. We focus on our recent work using MSOT to image BAT in resting and activated states both in mice and humans.

High resolution volumetric imaging of primary and secondary tumor spheroids using multi-angle Light Sheet Fluorescence Microscopy (LSFM).

Breast cancer and Glioblastoma brain cancer are severe malignancies with poor prognosis. In this study, primary Glioblastoma and secondary breast cancer spheroids are formed and treated with the well-known Temozolomide and Doxorubicin chemotherapeutics, respectively. High resolution imaging of both primary and secondary cancer cell spheroids is possible using a custom multi-angle Light Sheet Fluorescence Microscope. Such a technique is successful in realizing preclinical drug screening, while enables the discrimination among physiologic tumor parameters.

Demonstrating Improved Multiple Transport-Mean-Free-Path Imaging Capabilities of Light Sheet Microscopy in the Quantification of Fluorescence Dynamics.

Optical microscopy constitutes, one of the most fundamental paradigms for the understanding of complex biological mechanisms in the whole-organism and live-tissue context. Novel imaging techniques such as light sheet fluorescence microscopy (LSFM) and optical projection tomography (OPT) combined with phase-retrieval algorithms (PRT) can produce highly resolved 3D images in multiple transport-mean-free-path scales. Our study aims to exemplify the microscopic capabilities of LSFM when imaging protein dynamics in Caenorhabditis elegans and the distribution of necrotic cells in cancer cell spheroids. To this end, we apply LSFM to quantify the spatio-temporal localization of the GFP-tagged aging and stress response factor DAF-16/FOXO in transgenic C. elegans. Our analysis reveals a linear nuclear localization of DAF-16::GFP across tissues in response to heat stress, using a system that outperforms confocal scanning fluorescent microscopy in imaging speed, 3D resolution and reduced photo-toxicity. Furthermore, we present how PRT can improve the depth-to-resolution-ratio when applied to image the far-red fluorescent dye DRAQ7 which stains dead cells in a T47D cancer cell spheroid recorded with a customized OPT/LSFM system. Our studies demonstrate that LSFM combined with our novel approaches enables higher resolution and more accurate 3D quantification than previously applied technologies, proving its advance as new gold standard for fluorescence microscopy.

Optical projection tomography via phase retrieval algorithms.

We describe a computational method for accurate, quantitative tomographic reconstructions in Optical Projection Tomography, based on phase retrieval algorithms. Our method overcomes limitations imposed by light scattering in opaque tissue samples under the memory effect regime, as well as reduces artifacts due to mechanical movements, misalignments or vibrations. We make use of Gerchberg-Saxton algorithms, calculating first the autocorrelation of the object and then retrieving the associated phase under four numerically simulated measurement conditions. By approaching the task in such a way, we avoid the projection alignment procedure, exploiting the fact that the autocorrelation sinogram is always aligned and centered. We thus propose two new, projection-based, tomographic imaging flowcharts that allow registration-free imaging of opaque biological specimens and unlock three-dimensional tomographic imaging of hidden objects. Two main reconstruction approaches are discussed in the text, focusing on their efficiency in the tomographic retrieval and discussing their applicability under four different numerical experiments.

Phase-Retrieved Tomography enables Mesoscopic imaging of Opaque Tumor Spheroids.

We present a new Phase-Retrieved Tomography (PRT) method to radically improve mesoscopic imaging at regimes beyond one transport mean-free-path and achieve high resolution, uniformly throughout the volume of opaque samples. The method exploits multi-view acquisition in a hybrid Selective Plane Illumination Microscope (SPIM) and Optical Projection Tomography (OPT) setup and a three-dimensional Gerchberg-Saxton phase-retrieval algorithm applied in 3D through the autocorrelation sinogram. We have successfully applied this innovative protocol to image optically dense 3D cell cultures in the form of tumor spheroids, highly versatile models to study cancer behavior and response to chemotherapy. We have thus achieved a significant improvement of resolution in depths not yet accessible with the currently used methods in SPIM/OPT, while overcoming all registration and alignment problems inherent to these techniques.

Multispectral Optoacoustic Tomography (MSOT) of Human Breast Cancer.

Purpose: In a pilot study, we introduce fast handheld multispectral optoacoustic tomography (MSOT) of the breast at 28 wavelengths, aiming to identify high-resolution optoacoustic (photoacoustic) patterns of breast cancer and noncancerous breast tissue.Experimental Design: We imaged 10 female patients ages 48-81 years with malignant nonspecific breast cancer or invasive lobular carcinoma. Three healthy volunteers ages 31-36 years were also imaged. Fast-MSOT was based on unique single-frame-per-pulse (SFPP) image acquisition employed to improve the accuracy of spectral differentiation over using a small number of wavelengths. Breast tissue was illuminated at the 700-970 nm spectral range over 0.56 seconds total scan time. MSOT data were guided by ultrasonography and X-ray mammography or MRI.Results: The extended spectral range allowed the computation of oxygenated hemoglobin (HBO2), deoxygenated hemoglobin (HB), total blood volume (TBV), lipid, and water contributions, allowing first insights into in vivo high-resolution breast tissue MSOT cancer patterns. TBV and Hb/HBO2 images resolved marked differences between cancer and control tissue, manifested as a vessel-rich tumor periphery with highly heterogeneous spatial appearance compared with healthy tissue. We observe significant TBV variations between different tumors and between tumors over healthy tissues. Water and fat lipid layers appear disrupted in cancer versus healthy tissue; however, offer weaker contrast compared with TBV images.Conclusions: In contrast to optical methods, MSOT resolves physiologic cancer features with high resolution and revealed patterns not offered by other radiologic modalities. The new features relate to personalized and precision medicine potential. Clin Cancer Res; 23(22); 6912-22. ©2017 AACR.

A hybrid discrete-continuous model of in vitro spheroid tumor growth and drug response.

Anti-cancer therapy efficacy in solid tumors mainly depends on drug transportation through the vasculature system and the extracellular matrix, on diffusion gradients and clonal heterogeneity within the tumor mass, as well as on the responses of the individual tumor cells to drugs and their interactions with each other and their local microenvironment. In this work, we develop a mathematical predictive model for tumor growth and drug response based on 3D spheroids experiments that possess several in vivo features of tumors and are considered better for drug screening. The model takes into account the diffusion gradients of both oxygen and drug through the tumor volume, describes the tumor population at cell level and assumes a simple underlying cellular dose-response curve that is translated to a cell death probability. The model shows that although the endpoint tumor regression can be well approximated, the effects of the drug on cell fate necessitate a more sophisticated model to explain the temporal evolution of tumor regression and more quantitative information regarding the number and topology of dead and living cells, which is highly important for in vivo clinical relevant predictions. The model is built in a way that can be constrained by experimentally derived set of parameters and is capable of accommodating cell heterogeneity, sub-cellular regulatory mechanisms and drug-induced signaling cascades, as well as additional mechanisms of adapted resistance.

Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells.

The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.