A genetically encoded near-infrared fluorescent calcium ion indicator.

We report an intensiometric, near-infrared fluorescent, genetically encoded calcium ion (Ca2+) indicator (GECI) with excitation and emission maxima at 678 and 704 nm, respectively. This GECI, designated NIR-GECO1, enables imaging of Ca2+ transients in cultured mammalian cells and brain tissue with sensitivity comparable to that of currently available visible-wavelength GECIs. We demonstrate that NIR-GECO1 opens up new vistas for multicolor Ca2+ imaging in combination with other optogenetic indicators and actuators.

Hybrid system for in vivo epifluorescence and 4D optoacoustic imaging.

Epifluorescence imaging is widely used in cell and molecular biology due to its excellent sensitivity, contrast, and ease of implementation. Optoacoustic imaging has been shown to deliver a highly complementary and unique set of capabilities for biological discovery, such as high spatial resolution in noninvasive deep tissue observations, fast volumetric imaging capacity, and spectrally enriched contrast. In this Letter, we report on a hybrid system combining planar fluorescence and real-time volumetric four-dimensional optoacoustic imaging by means of a fiberscope integrated within a handheld hemispherical ultrasound detection array. The in vivo imaging performance is demonstrated by non-invasive visualization of fast contrast agent perfusion through the mouse brain. The proposed synergistic combination of fluorescence and optoacoustic imaging can benefit numerous studies looking at multi-scale in vivo dynamics, such as functional neuroimaging, visualization of organ perfusion and contrast agent uptake, cell tracking, and pharmacokinetic and bio-distribution analysis.

Functional optoacoustic neuro-tomography of calcium fluxes in adult zebrafish brain in vivo.

Genetically-encoded calcium indicators (GECIs) have revolutionized neuroimaging by enabling mapping of the activity of entire neuronal populations in vivo. Visualization of these powerful activity sensors has to date been limited to depth-restricted microscopic studies due to intense light scattering in the brain. We demonstrate, for the first time, in vivo real-time volumetric optoacoustic monitoring of calcium transients in adult transgenic zebrafish expressing the GCaMP5G calcium indicator. Fast changes in optoacoustic traces associated with GCaMP5G activity were detectable in the presence of other strongly absorbing endogenous chromophores, such as hemoglobin. The new functional optoacoustic neuroimaging method can visualize neural activity at penetration depths and spatio-temporal resolution scales not covered with the existing neuroimaging techniques.

Magnetic resonance and optical imaging probes for NMDA receptors on the cell surface of neurons: synthesis and evaluation in cellulo.

A second generation of N-methyl-d-aspartate (NMDA) receptor-targeted MRI contrast agents has been synthesised and evaluated in cellulo, based on established bicyclic NMDA receptor antagonists. Their use as responsive MR imaging probes has been evaluated in suspensions of NSC-34 cells, and one agent exhibited significant enhancements in measured longitudinal and transverse water proton relaxation rates (19 and 38% respectively; 3 T, 298 K). A biotin derivative of the lead compound was prepared and the specificity and reversibility of binding to the NMDA cell surface receptors demonstrated using confocal laser scanning microscopy. Competitive and reversible binding of glutamate to the receptors was also visualised, suggesting that the receptor-targeted approach may allow MRI to be used to monitor neuronal events associated with modulation of local glutamate concentrations.

Multifunctional silica nanoparticles for optical and magnetic resonance imaging.

The surface of spherical, nonporous silica nanoparticles (SiO2-NPs) was modified with gadolinium (Gd) complexes, fluorophores, and cell-penetrating peptides to achieve multifunctionality on a single particle. The Gd surface concentrations were 9-16 µmol/g resulting in nanomaterials with high local longitudinal and transversal relaxivities (~1×10(5) and ~5×10(5) /mm/s/NP, respectively). Rapid cellular uptake was observed in vitro; however, larger extracellular agglomerates were also formed. In vivo administration revealed a fast distribution throughout the body followed by a nearly complete disappearance of fluorescence in all organs except the lungs, liver, and spleen after 24 h. Such NPs have the potential to serve as efficient multimodal probes in molecular imaging.

Comparative in vitro studies of MR imaging probes for metabotropic glutamate subtype-5 receptor targeting.

A series of magnetic resonance imaging probes has been evaluated to target selectively the metabotropic glutamate receptor subtype 5 (mGluR5). Eight imaging probes based on the contrast agent [Gd·DOTA], previously derived by linking it to a series of specific and selective mGluR5 antagonists, have been extensively tested for their functionality in vitro. The Nuclear Magnetic Relaxation Dispersion (NMRD) profiles of selected probes have been examined via field-cycling relaxometry in the presence and absence of a model protein. The properties of the targeted contrast agents were evaluated using a primary astrocyte model, as these cells mimic the in vivo situation effectively. The probes were non-toxic (up to 200 µM) to these mGluR5 expressing primary cells. Cellular proton longitudinal relaxation rate enhancements of up to 35% were observed by MRI at 200 µM of probe concentration. The antagonistic effect of all compounds was tested using an assay measuring changes of intracellular calcium levels. Furthermore, treatment at two different temperatures (4 °C vs. 37 °C) and of an mGluR5-negative cell line provided further insight into the selectivity and specificity of these probes towards cell surface mGluR5. Finally, two out of eight probes demonstrated an antagonistic effect as well as significant enhancement of receptor mediated cellular relaxation rates, strongly suggesting that they would be viable probes for the mapping of mGluR5 by MRI in vivo.

Hepatocellular apoptosis in mice is associated with early upregulation of mitochondrial glucose metabolism.

Hepatocyte death due to apoptosis is a hallmark of almost every liver disease. Manipulation of cell death regulatory steps during the apoptotic process is therefore an obvious goal of biomedical research. To clarify whether metabolic changes occur prior to the characteristic apoptotic events, we used ex vivo multinuclear NMR-spectroscopy to study metabolic pathways of [U-(13)C]glucose in mouse liver during Fas-induced apoptosis. We addressed whether these changes could be associated with protection against apoptosis afforded by Epidermal Growth Factor (EGF). Our results show that serum alanine and aspartate aminotransferase levels, caspase-3 activity, BID cleavage and changes in cellular energy stores were not observed before 3 h following anti-Fas injection. However, as early as 45 min after anti-Fas treatment, we observed upregulation of carbon entry (i.e. flux) from glucose into the Krebs-cycle via pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) (up to 139% and 123% of controls, respectively, P < 0.001). This was associated with increased glutathione synthesis. EGF treatment significantly attenuated Fas-induced apoptosis, liver injury and the late decrease in energy stores, as well as the early fluxes through PDH and PC which were comparable to untreated controls. Using ex vivo multinuclear NMR-spectroscopic analysis, we have shown that Fas receptor activation in mouse liver time-dependently affects specific metabolic pathways of glucose. These early upregulations in glucose metabolic pathways occur prior to any visible signs of apoptosis and may have the potential to contribute to the initiation of apoptosis by maintaining mitochondrial energy production and cellular glutathione stores.

CyLoP-1: a novel cysteine-rich cell-penetrating peptide for cytosolic delivery of cargoes.

Cell-penetrating peptides (CPPs) may have impli-cations in biomedical sciences by improving the delivery of a wide variety of drugs through the membrane barrier. CPPs are generally taken up by endocytotic pathways, and vesicular encapsulation is a limiting factor in the area of intracellular targeting. A novel, cationic cysteine-rich CPP, CyLoP-1, has been developed exhibiting distinguished diffused cytosolic distribution along with endosomal uptake at low micromolar concentrations. Comparative uptake analysis with known CPPs showed CyLoP-1 as a promising delivery vector to access the cytosol in a variety of cell types. In addition to the positively charged residues, the presence of cysteines and tryptophans proved to be essential to maintain its functionality. Also, the oxidation status of the cysteines played an important role for the uptake efficiency of CyLoP-1, with the disulfide-containing form being more effective. The distinct feature of CyLoP-1 to enter the cytosol was further explored by the covalent attachment of cargoes of different nature and sizes. In particular, induction of caspase-3 activity (indicating apoptosis) by a CyLoP-1-SmacN7 conjugate proved successful delivery of the pro-apoptotic cargo to its site of action in the cytosol. Efficient intracellular delivery into the entire cytosol already at low micromolar concentrations makes CyLoP-1 a promising candidate for cytosolic delivery of cargoes of small sizes. Thus, this peptide might prove to be useful for efficient transmembrane delivery of agents directed to cytosolic targets.

Immunosuppressant neurotoxicity in rat brain models: oxidative stress and cellular metabolism.

Coadministration of the calcineurin inhibitor cyclosporine (CsA) and the mTOR inhibitors sirolimus (SRL) or everolimus (RAD) increases the efficacy of immunosuppression after organ transplantation. Neurotoxicity of CsA is a major clinical problem. Our goal was to assess the effects of CsA, SRL, and RAD on brain cell metabolism. The studies included the comparison of immunosuppressant-mediated effects on glucose metabolism, energy production, and reactive oxygen species (ROS) formation in perfused rat brain slices, primary rat astrocytes, and C6 glioma cells. In brain slices and astrocytes, CsA inhibited Krebs cycle metabolism, while activating anaerobic glycolysis, most likely to compensate for the inhibition of mitochondrial energy production. SRL and RAD inhibited cytosolic glycolysis but did not cause changes in mitochondrial energy production. CsA + SRL inhibited Krebs cycle and glycolysis, thus reducing the ability of the cell to compensate for the negative effects of CsA on mitochondrial nucleoside triphosphate synthesis. In contrast to SRL at the concentrations tested, RAD reduced the CsA-induced ROS formation and antagonized CsA-induced effects on glucose and energy metabolism. Surprisingly, in C6 cells, SRL and RAD exposure resulted in high ROS concentrations without significant impairment of cell metabolism. Our results suggested that SRL enhances CsA-induced ROS formation and negative metabolic effects in brain cells, while RAD seems to antagonize the CsA effects. However, the three models showed different metabolic responses when challenged with the study drugs. In contrast to SRL, RAD enhances ROS formation in C6 glioma cells but has only minor effects on normal rat brain tissue.

Macrocyclic Gd3+ chelates attached to a silsesquioxane core as potential magnetic resonance imaging contrast agents: synthesis, physicochemical characterization, and stability studies.

Two macrocyclic ligands, 1,4,7,10-tetraazacyclododecane-1-glutaric-4,7,10-triacetic acid (H(5)DOTAGA) and the novel 1,4,7,10-tetraazacyclododecane-1-(4-(carboxymethyl)benzoic)-4,7,10-triacetic acid (H(5)DOTABA), were prepared and their lanthanide complexes (Ln = Gd(3+), Y(3+)) attached to an amino-functionalized T(8)-silsesquioxane. The novel compounds Gadoxane G (GG) and Gadoxane B (GB) possess eight monohydrated lanthanide complexes each, as evidenced by multinuclear ((1)H, (13)C, (29)Si) NMR spectroscopy and high resolution mass spectrometry (HR-MS). Pulsed-field gradient spin echo (PGSE) diffusion (1)H NMR measurements revealed hydrodynamic radii of 1.44 nm and global rotational correlation times of about 3.35 ns for both compounds. With regard to potential MRI contrast agent applications, a variable-temperature (17)O NMR and (1)H nuclear magnetic relaxation dispersion (NMRD) study was carried out on aqueous solutions of the gadolinium(III) complexes of the Gadoxanes and the corresponding monomeric ligands to yield relevant physicochemical properties. The water exchange rates of the inner-sphere water molecules are all very similar (k(ex)(298) between (5.3 +/- 0.5) x 10(6) s(-1) and (5.9 +/- 0.3) x 10(6) s(-1)) and only slightly higher than that reported for the gadolinium(III) complex of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (H(4)DOTA) (k(ex)(298) = 4.1 x 10(6) s(-1)). Despite their almost identical size and their similar water exchange rates, GB shows a significantly higher longitudinal relaxivity than GG over nearly the whole range of magnetic fields (e.g., 17.1 mM(-1) s(-1) for GB and 12.1 mM(-1) s(-1) for GG at 20 MHz and 25 degrees C). This difference arises from their different local rotational correlation times (tau(lR)(298) = 240 +/- 10 ps and 380 +/- 20 ps, respectively), because of the higher rigidity of the phenyl ring of GB as compared to the ethylene spacer of GG. A crucial feature of these novel compounds is the lability of the silsesquioxane core in aqueous media. The hydrolysis of the Si-O-Si moieties was investigated by (29)Si NMR and PGSE diffusion (1)H NMR spectroscopy, electrospray ionization mass spectrometry (ESI-MS), as well as by relaxivity measurements. Although frozen aqueous solutions (pH 7.0) of GG and GB can be stored at -28 degrees C for at least 10 months without any decomposition, with increasing temperature and pH the hydrolysis of the silsesquioxane core was observed (e.g., t(1/2) = 15 h at pH 7.4 and 55 min at pH 8.1 for GG at 37 degrees C). No change in relaxivity was detected within the first 3 h, since the hydrolysis of the initial Si-O-Si moieties has no influence on the rotational correlation time. However, the further hydrolysis of the silsesquioxane core leads to smaller fragments and therefore to a decrease in relaxivity.

Publisher Correction: Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Intravital optoacoustic and ultrasound bio-microscopy reveal radiation-inhibited skull angiogenesis.

Angiogenesis is critical in bone development and growth. Dense, large-scale, and multi-layered vascular networks formed by thin-walled sinusoidal vessels perfuse the plate bones and play an important role in bone repair. Yet, the intricate functional morphology of skull microvasculature remains poorly understood as it is difficult to visualize using existing intravital microscopy techniques. Here we introduced an intravital, fully-transcranial imaging approach based on hybrid optoacoustic and ultrasound bio-microscopy for large-scale observations and quantitative analysis of the vascular morphology, angiogenesis, vessel remodeling, and subsurface roughness in murine skulls. Our approach revealed radiation-inhibited angiogenesis in the skull bone. We also observed previously undocumented sinusoidal vascular networks spanning the entire skullcap, thus opening new vistas for studying the complex interactions between calvarial, pial, and cortical vascular systems.

Altered fatty acid metabolism and composition in cultured astrocytes under hyperammonemic conditions.

In vitro 1H- and 13C-NMR spectroscopy was used to investigate the effect of ammonia on fatty acid synthesis and composition in cultured astrocytes. Cells were incubated 3 and 24 h with 5 mM ammonia in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. An increase of de novo synthesized fatty acids and the glycerol subunit of lipids was observed after 3 h treatment with ammonia (35% and 40% over control, respectively), the initial time point examined. Both parameters further increased significantly to 85% and 60% over control after 24 h ammonia treatment. Three hours incubation with ammonia increased the synthesis of diacylglycerides, while formation of triacylglycerides was decreased (40% over and 15% under control, respectively). The degradation of fatty acids was not affected by ammonia treatment. Furthermore, ammonia caused alterations in the composition of fatty acids, e.g. increased mono- and decreased polyunsaturated fatty acids (85% over and 15% under control concentrations, respectively). The decrease of polyunsaturated fatty acids was even more pronounced in isolated astrocytic mitochondria (39% lower than controls). Our results suggest ammonia-induced abnormalities in astrocytic membranes, which may be related to astrocytic mitochondrial dysfunction in hyperammonemic states. Most of the observed effects of ammonia on fatty acid synthesis and composition were ameliorated when glutamine synthetase was inhibited by methionine sulfoximine, supporting a pathological role of glutamine in ammonia toxicity. This study further emphasizes the importance of investigating the relative contribution of exogenous ammonia, effects of glutamine and of glutamine-derived ammonia on astrocytes and astrocytic mitochondria.

Volumetric Optoacoustic Imaging Unveils High-Resolution Patterns of Acute and Cyclic Hypoxia in a Murine Model of Breast Cancer.

Mapping tumor heterogeneity and hypoxia within a living intact organism is essential for understanding the processes involved in cancer progression and assessing long-term responses to therapies. Efficient investigations into tumor hypoxia mechanisms have been hindered by the lack of intravital imaging tools capable of multiparametric probing of entire solid tumors with high spatial and temporal resolution. Here, we exploit volumetric multispectral optoacoustic tomography (vMSOT) for accurate, label-free delineation of tumor heterogeneity and dynamic oxygenation behavior. Mice bearing orthotopic MDA-MB-231 breast cancer xenografts were imaged noninvasively during rest and oxygen stress challenge, attaining time-lapse three-dimensional oxygenation maps across entire tumors with 100 µm spatial resolution. Volumetric quantification of the hypoxic fraction rendered values of 3.9% to 21.2%, whereas the oxygen saturation (sO2) rate declined at 1.7% to 2.3% per mm in all tumors when approaching their core. Three distinct functional areas (the rim, hypoxic, and normoxic cores) were clearly discernible based on spatial sO2 profiles and responses to oxygen challenge. Notably, although sO2 readings were responsive to the challenge, deoxyhemoglobin (HbR) trends exhibited little to no variations in all mice. Dynamic analysis further revealed the presence of cyclic hypoxia patterns with a 21% average discrepancy between cyclic fractions assessed via sO2 (42.2% ± 17.3%) and HbR fluctuations (63% ± 14.1%) within the hypoxic core. These findings corroborate the strong potential of vMSOT for advancing preclinical imaging of cancer and informing clinical decisions on therapeutic interventions. SIGNIFICANCE: vMSOT provides quantitative measures of volumetric hypoxic fraction and cyclic hypoxia in a label-free and noninvasive manner, providing new readouts to aid tumor staging and treatment decision making. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/79/18/4767/F1.large.jpg.See related commentary by Klibanov and Hu, p. 4577.

Non-invasive determination of murine placental and foetal functional parameters with multispectral optoacoustic tomography.

Despite the importance of placental function in embryonic development, it remains poorly understood and challenging to characterize, primarily due to the lack of non-invasive imaging tools capable of monitoring placental and foetal oxygenation and perfusion parameters during pregnancy. We developed an optoacoustic tomography approach for real-time imaging through entire ~4 cm cross-sections of pregnant mice. Functional changes in both maternal and embryo regions were studied at different gestation days when subjected to an oxygen breathing challenge and perfusion with indocyanine green. Structural phenotyping of the cross-sectional scans highlighted different internal organs, whereas multi-wavelength acquisitions enabled non-invasive label-free spectroscopic assessment of blood-oxygenation parameters in foeto-placental regions, rendering a strong correlation with the amount of oxygen administered. Likewise, the placental function in protecting the embryo from extrinsically administered agents was substantiated. The proposed methodology may potentially further serve as a probing mechanism to appraise embryo development during pregnancy in the clinical setting.

Rapid volumetric optoacoustic imaging of neural dynamics across the mouse brain.

Efforts to scale neuroimaging towards the direct visualization of mammalian brain-wide neuronal activity have faced major challenges. Although high-resolution optical imaging of the whole brain in small animals has been achieved ex vivo, the real-time and direct monitoring of large-scale neuronal activity remains difficult, owing to the performance gap between localized, largely invasive, optical microscopy of rapid, cellular-resolved neuronal activity and whole-brain macroscopy of slow haemodynamics and metabolism. Here, we demonstrate both ex vivo and non-invasive in vivo functional optoacoustic (OA) neuroimaging of mice expressing the genetically encoded calcium indicator GCaMP6f. The approach offers rapid, high-resolution three-dimensional snapshots of whole-brain neuronal activity maps using single OA excitations, and of stimulus-evoked slow haemodynamics and fast calcium activity in the presence of strong haemoglobin background absorption. By providing direct neuroimaging at depths and spatiotemporal resolutions superior to optical fluorescence imaging, functional OA neuroimaging bridges the gap between functional microscopy and whole-brain macroscopy.

Uniform light delivery in volumetric optoacoustic tomography.

Accurate image reconstruction in volumetric optoacoustic tomography implies the efficient generation and collection of ultrasound signals around the imaged object. Non-uniform delivery of the excitation light is a common problem in optoacoustic imaging often leading to a diminished field of view, limited dynamic range and penetration, as well as impaired quantification abilities. Presented here is an optimized illumination concept for volumetric tomography that utilizes additive manufacturing via 3D printing in combination with custom-made optical fiber illumination. The custom-designed sample chamber ensures convenient access to the imaged object along with accurate positioning of the sample and a matrix array ultrasound transducer used for collection of the volumetric image data. Ray tracing is employed to optimize the positioning of the individual fibers in the chamber. Homogeneity of the generated light excitation field was confirmed in tissue-mimicking agar spheres. Applicability of the system to image entire mouse organs ex vivo has been showcased. The new approach showed a clear advantage over conventional, single-sided illumination strategies by eliminating the need to correct for illumination variances and resulting in enhancement of the effective field of view, greater penetration depth and significant improvements in the overall image quality.

Isolated Murine Brain Model for Large-Scale Optoacoustic Calcium Imaging.

Real-time visualization of large-scale neural dynamics in whole mammalian brains is hindered with existing neuroimaging methods having limited capacity when it comes to imaging large tissue volumes at high speeds. Optoacoustic imaging has been shown to be capable of real-time three-dimensional imaging of multiple cerebral hemodynamic parameters in rodents. However, optoacoustic imaging of calcium activity deep within the mammalian brain is hampered by strong blood absorption in the visible light spectrum as well as a lack of activity labels excitable in the near-infrared window. We have developed and validated an isolated whole mouse brain preparation labeled with genetically encoded calcium indicator GCaMP6f, which can closely resemble in vivo conditions. An optoacoustic imaging system coupled to a superfusion system was further designed and used for rapid volumetric monitoring of stimulus-evoked calcium dynamics in the brain. These new imaging setup and isolated preparation's protocols and characteristics are described here in detail. Our new technique captures calcium fluxes as true three-dimensional information across the entire brain with temporal resolution of 10 ms and spatial resolution of 150 µm, thus enabling large-scale neural recording at penetration depths and spatio-temporal resolution scales not covered with any existing neuroimaging techniques.

Concurrent fluorescence and volumetric optoacoustic tomography of nanoagent perfusion and bio-distribution in solid tumors.

Intravenously administered liposomes and other nano-sized particles are known to passively accumulate in solid tumors via the enhanced permeability and retention (EPR) effect, which is extensively explored toward the improvement of diagnosis and drug delivery in oncology. Agent extravasation into tumors is often hampered by the mononuclear phagocytic and renal systems, which sequester and/or eliminate most of the nanoparticles from the body. Dynamic imaging of the tumor microcirculation and bolus perfusion can thus facilitate optimization of the nanoparticle delivery. When it comes to non-invasive visualization of rapid biological dynamics in whole tumors, the currently available pre-clinical imaging modalities are commonly limited by shallow penetration, lack of suitable contrast or otherwise insufficient spatial or temporal resolution. Herein, we demonstrate the unique capabilities of a combined epi-fluorescence and optoacoustic tomography (FLOT) system for characterizing contrast agent dynamics in orthotopic breast tumors in mice. A liposomal indocyanine green (Lipo-ICG) preparation was administered intravenously with the time-lapse data continuously acquired during and after the injection procedure. In addition to the highly sensitive detection of the fluorescence agent by the epi-fluorescence modality, the volumetric multi-spectral optoacoustic tomography readings further enabled resolving deep-seated vascular structures with high spatial resolution and hence provided accurate readings of the dynamic bio-distribution of nanoparticles in the entire tumor in 3D. The synergetic combination of the two modalities can become a powerful tool in cancer research and potentially aid the diagnosis, staging and treatment guidance of certain types of cancer in the clinical setting.

Observation of Guided Acoustic Waves in a Human Skull.

Human skull poses a significant barrier for the propagation of ultrasound waves. Development of methods enabling more efficient ultrasound transmission into and from the brain is therefore critical for the advancement of ultrasound-mediated transcranial imaging or actuation techniques. We report on the first observation of guided acoustic waves in the near field of an ex vivo human skull specimen in the frequency range between 0.2 and 1.5MHz. In contrast to what was previously observed for guided wave propagation in thin rodent skulls, the guided wave observed in a higher-frequency regime corresponds to a quasi-Rayleigh wave, confined mostly to the cortical bone layer. The newly discovered near-field properties of the human skull are expected to facilitate the development of more efficient diagnostic and therapeutic techniques based on transcranial ultrasound.