Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria.

Bacteriophages are potent therapeutics against biohazardous bacteria, which rapidly develop multidrug resistance. However, routine administration of phage therapy is hampered by a lack of rapid production, safe bioengineering, and detailed characterization of phages. Thus, we demonstrate a comprehensive cell-free platform for personalized production, transient engineering, and proteomic characterization of a broad spectrum of phages. Using mass spectrometry, we validated hypothetical and non-structural proteins and could also monitor the protein expression during phage assembly. Notably, a few microliters of a one-pot reaction produced effective doses of phages against enteroaggregative Escherichia coli (EAEC), Yersinia pestis, and Klebsiella pneumoniae. By co-expressing suitable host factors, we could extend the range of cell-free production to phages targeting gram-positive bacteria. We further introduce a non-genomic phage engineering method, which adds functionalities for only one replication cycle. In summary, we expect this cell-free methodology to foster reverse and forward phage engineering and customized production of clinical-grade bacteriophages.

Genetically Encoded Self-Assembling Iron Oxide Nanoparticles as a Possible Platform for Cancer-Cell Tracking.

The study of growth and possible metastasis in animal models of tumors would benefit from reliable cell labels for noninvasive whole-organism imaging techniques such as magnetic resonance imaging. Genetically encoded cell-tracking reporters have the advantage that they are contrast-selective for viable cells with intact protein expression machinery. Besides, these reporters do not suffer from dilution during cell division. Encapsulins, which are bacterial protein nanocompartments, can serve as genetically controlled labels for multimodal detection of cells. Such nanocompartments can host various guest molecules inside their lumen. These include, for example, fluorescent proteins or enzymes with ferroxidase activity leading to biomineralization of iron oxide inside the encapsulin nanoshell. The aim of this work was to implement heterologous expression of encapsulin systems from Quasibacillus thermotolerans using the fluorescent reporter protein mScarlet-I and ferroxidase IMEF in the human hepatocellular carcinoma cell line HepG2. The successful expression of self-assembled encapsulin nanocompartments with functional cargo proteins was confirmed by fluorescence microscopy and transmission electron microscopy. Also, coexpression of encapsulin nanoshells, ferroxidase cargo, and iron transporter led to an increase in T2-weighted contrast in magnetic resonance imaging of HepG2 cells. The results demonstrate that the encapsulin cargo system from Q. thermotolerans may be suitable for multimodal imaging of cancer cells and could contribute to further in vitro and in vivo studies.

Structural perspective on revealing heat dissipation behavior of CoFe2O4-Pd nanohybrids: great promise for magnetic fluid hyperthermia.

Loss mechanisms in fluid heating of cobalt ferrite (CFO) nanoparticles and CFO-Pd heterodimer colloidal suspensions are investigated as a function of particle size, fluid concentration and magnetic field amplitude. The specific absorption rate (SAR) is found to vary with increasing particle size due to a change in dominant heating mechanism from susceptibility to hysteresis and frictional loss. The maximum SAR is obtained for particle diameters of 11-15 nm as a result of synergistic contributions of susceptibility loss, including Néel and Brownian relaxation and especially hysteresis loss, thereby validating the applicability of linear response theory to superparamagnetic CFO nanoparticles. Our results show that the ferrofluid concentration and magnetic field amplitude alter interparticle interactions and associated heating efficiency. The SAR of the CFO nanoparticles could be maximized by adjusting the synthesis parameters. Despite the paramagnetic properties of individual palladium nanoparticles, CFO-Pd heterodimer suspensions were observed to have surprisingly improved magnetization as well as SAR values, when compared with CFO ferrofluids. This difference is attributed to interfacial interactions between the magnetic moments of paramagnetic Pd and superparamagnetic/ferrimagnetic CFO. SAR values measured from CFO-Pd heterodimer suspensions were found to be 47-52 W gFerrite-1, which is up to a factor of two higher than the SAR values of commercially available ferrofluids, demonstrating their potential as efficient heat mediators. Our results provide insight into the utilization of CFO-Pd heterodimer suspensions as potential nanoplatforms for diagnostic and therapeutic biomedical applications, e.g., in cancer hyperthermia, cryopreserved tissue warming, thermoablative therapy, drug delivery and bioimaging.

Artifact-free deconvolution in light field microscopy.

The sampling patterns of the light field microscope (LFM) are highly depth-dependent, which implies non-uniform recoverable lateral resolution across depth. Moreover, reconstructions using state-of-the-art approaches suffer from strong artifacts at axial ranges, where the LFM samples the light field at a coarse rate. In this work, we analyze the sampling patterns of the LFM, and introduce a flexible light field point spread function model (LFPSF) to cope with arbitrary LFM designs. We then propose a novel aliasing-aware deconvolution scheme to address the sampling artifacts. We demonstrate the high potential of the proposed method on real experimental data.

Iron-Sequestering Nanocompartments as Multiplexed Electron Microscopy Gene Reporters.

Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of ∼43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.

Whole-Cell Photoacoustic Sensor Based on Pigment Relocalization.

Photoacoustic (optoacoustic) imaging can extract molecular information with deeper tissue penetration than possible by fluorescence microscopy techniques. However, there is currently still a lack of robust genetically controlled contrast agents and molecular sensors that can dynamically detect biological analytes of interest with photoacoustics. In a biomimetic approach, we took inspiration from cuttlefish who can change their color by relocalizing pigment-filled organelles in so-called chromatophore cells under neurohumoral control. Analogously, we tested the use of melanophore cells from Xenopus laevis, containing compartments (melanosomes) filled with strongly absorbing melanin, as whole-cell sensors for optoacoustic imaging. Our results show that pigment relocalization in these cells, which is dependent on binding of a ligand of interest to a specific G protein-coupled receptor (GPCR), can be monitored in vitro and in vivo using photoacoustic mesoscopy. In addition to changes in the photoacoustic signal amplitudes, we could furthermore detect the melanosome aggregation process by a change in the frequency content of the photoacoustic signals. Using bioinspired engineering, we thus introduce a photoacoustic pigment relocalization sensor (PaPiReS) for molecular photoacoustic imaging of GPCR-mediated signaling molecules.

Author Correction: Zebrafish and medaka offer insights into the neurobehavioral correlates of vertebrate magnetoreception.

Text for Correction.

Bacterial encapsulins as orthogonal compartments for mammalian cell engineering.

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.

Zebrafish and medaka offer insights into the neurobehavioral correlates of vertebrate magnetoreception.

An impediment to a mechanistic understanding of how some species sense the geomagnetic field ("magnetoreception") is the lack of vertebrate genetic models that exhibit well-characterized magnetoreceptive behavior and are amenable to whole-brain analysis. We investigated the genetic model organisms zebrafish and medaka, whose young stages are transparent and optically accessible. In an unfamiliar environment, adult fish orient according to the directional change of a magnetic field even in darkness. To enable experiments also in juveniles, we applied slowly oscillating magnetic fields, aimed at generating conflicting sensory inputs during exploratory behavior. Medaka (but not zebrafish) increase their locomotor activity in this assay. Complementary brain  activity mapping reveals neuronal activation in the lateral hindbrain during magnetic stimulation. These comparative data support magnetoreception in teleosts, provide evidence for a light-independent mechanism, and demonstrate the usefulness of zebrafish and medaka as genetic vertebrate models for studying the biophysical and neuronal mechanisms underlying magnetoreception.

Calcium Sensor for Photoacoustic Imaging.

We introduce a selective and cell-permeable calcium sensor for photoacoustics (CaSPA), a versatile imaging technique that allows for fast volumetric mapping of photoabsorbing molecules with deep tissue penetration. To optimize for Ca2+-dependent photoacoustic signal changes, we synthesized a selective metallochromic sensor with high extinction coefficient, low quantum yield, and high photobleaching resistance. Micromolar concentrations of Ca2+ lead to a robust blueshift of the absorbance of CaSPA, which translated into an accompanying decrease of the peak photoacoustic signal. The acetoxymethyl esterified sensor variant was readily taken up by cells without toxic effects and thus allowed us for the first time to perform live imaging of Ca2+ fluxes in genetically unmodified cells and heart organoids as well as in zebrafish larval brain via combined fluorescence and photoacoustic imaging.

NeuBtracker-imaging neurobehavioral dynamics in freely behaving fish.

A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.

Microfluidic sorting of intrinsically magnetic cells under visual control.

Magnetic cell sorting provides a valuable complementary mechanism to fluorescent techniques, especially if its parameters can be fine-tuned. In addition, there has recently been growing interest in studying naturally occurring magnetic cells and genetic engineering of cells to render them magnetic in order to control molecular processes via magnetic fields. For such approaches, contamination-free magnetic separation is an essential capability. We here present a robust and tunable microfluidic sorting system in which magnetic gradients of up to 1700 T/m can be applied to cells flowing through a sorting channel by reversible magnetization of ferrofluids. Visual control of the sorting process allowed us to optimize sorting efficiencies for a large range of sizes and magnetic moments of cells. Using automated quantification based on imaging of fluorescent markers, we showed that macrophages containing phagocytosed magnetic nanoparticles, with cellular magnetic dipole moments on the order of 10 fAm2, could be sorted with an efficiency of 90 ± 1%. Furthermore, we successfully sorted intrinsically magnetic magnetotactic bacteria with magnetic moments of 0.1 fAm2. In distinction to column-based magnetic sorting devices, microfluidic systems can prevent sample contact with superparamagnetic material. This ensures contamination-free separation of naturally occurring or bioengineered magnetic cells and is essential for downstream characterization of their properties.

Reward magnitude tracking by neural populations in ventral striatum.

Evaluation of the magnitudes of intrinsically rewarding stimuli is essential for assigning value and guiding behavior. By combining parametric manipulation of a primary reward, medial forebrain bundle (MFB) microstimulation, with functional magnetic imaging (fMRI) in rodents, we delineated a broad network of structures activated by behaviorally characterized levels of rewarding stimulation. Correlation of psychometric behavioral measurements with fMRI response magnitudes revealed regions whose activity corresponded closely to the subjective magnitude of rewards. The largest and most reliable focus of reward magnitude tracking was observed in the shell region of the nucleus accumbens (NAc). Although the nonlinear nature of neurovascular coupling complicates interpretation of fMRI findings in precise neurophysiological terms, reward magnitude tracking was not observed in vascular compartments and could not be explained by saturation of region-specific hemodynamic responses. In addition, local pharmacological inactivation of NAc changed the profile of animals' responses to rewards of different magnitudes without altering mean reward response rates, further supporting a hypothesis that neural population activity in this region contributes to assessment of reward magnitudes.

Near-Infrared Photoacoustic Imaging Probe Responsive to Calcium.

Photoacoustic imaging (PAI) is an attractive imaging modality that can volumetrically map the distribution of photoabsorbing molecules with deeper tissue penetration than multiphoton microscopy. To enable dynamic sensing of divalent cations via PAI, we have engineered a new reversible near-infrared probe that is more sensitive to calcium as compared to other biologically relevant cations. The metallochromic compound showed a strong reduction of its peak absorbance at 765 nm upon addition of calcium ions that was translated into robust signal changes in photoacoustic images. Therefore, the heptamethine cyanine dye will be an attractive scaffold to create a series of metallochromic sensors for molecular PAI.

Hyperpolarized Multi-Metal 13C-Sensors for Magnetic Resonance Imaging.

We introduce hyperpolarizable 13C-labeled probes that identify multiple biologically important divalent metals via metal-specific chemical shifts. These features enable NMR measurements of calcium concentrations in human serum in the presence of magnesium. In addition, signal enhancement through dynamic nuclear polarization (DNP) increases the sensitivity of metal detection to afford measuring micromolar concentrations of calcium as well as simultaneous multi-metal detection by chemical shift imaging. The hyperpolarizable 13C-MRI sensors presented here enable sensitive NMR measurements and MR imaging of multiple divalent metals in opaque biological samples.

Magnetoacoustic Sensing of Magnetic Nanoparticles.

The interaction of magnetic nanoparticles and electromagnetic fields can be determined through electrical signal induction in coils due to magnetization. However, the direct measurement of instant electromagnetic energy absorption by magnetic nanoparticles, as it relates to particle characterization or magnetic hyperthermia studies, has not been possible so far. We introduce the theory of magnetoacoustics, predicting the existence of second harmonic pressure waves from magnetic nanoparticles due to energy absorption from continuously modulated alternating magnetic fields. We then describe the first magnetoacoustic system reported, based on a fiber-interferometer pressure detector, necessary for avoiding electric interference. The magnetoacoustic system confirmed the existence of previously unobserved second harmonic magnetoacoustic responses from solids, magnetic nanoparticles, and nanoparticle-loaded cells, exposed to continuous wave magnetic fields at different frequencies. We discuss how magnetoacoustic signals can be employed as a nanoparticle or magnetic field sensor for biomedical and environmental applications.

Functional optoacoustic neuro-tomography for scalable whole-brain monitoring of calcium indicators.

Non-invasive observation of spatiotemporal activity of large neural populations distributed over entire brains is a longstanding goal of neuroscience. We developed a volumetric multispectral optoacoustic tomography platform for imaging neural activation deep in scattering brains. It can record 100 volumetric frames per second across scalable fields of view ranging between 50 and 1000 mm3 with respective spatial resolution of 35-200 µm. Experiments performed in immobilized and freely swimming larvae and in adult zebrafish brains expressing the genetically encoded calcium indicator GCaMP5G demonstrate, for the first time, the fundamental ability to directly track neural dynamics using optoacoustics while overcoming the longstanding penetration barrier of optical imaging in scattering brains. The newly developed platform thus offers unprecedented capabilities for functional whole-brain observations of fast calcium dynamics; in combination with optoacoustics' well-established capacity for resolving vascular hemodynamics, it could open new vistas in the study of neural activity and neurovascular coupling in health and disease.

Light fluence normalization in turbid tissues via temporally unmixed multispectral optoacoustic tomography.

Discerning the accurate distribution of chromophores and biomarkers by means of optoacoustic imaging is commonly challenged by the highly heterogeneous excitation light patterns resulting from strong spatial variations of tissue scattering and absorption. Here we used the light-fluence dependent switching kinetics of reversibly switchable fluorescent proteins (RSFPs), in combination with real-time acquisition of volumetric multi-spectral optoacoustic data to correct for the light fluence distribution deep in scattering media. The new approach allows for dynamic fluence correction in time-resolved imaging, e.g., of moving organs, and can be extended to work with a large palette of available synthetic and genetically encoded photochromic substances for multiplexed wavelength-specific fluence normalization.

Volumetric tracking of migratory melanophores during zebrafish development by optoacoustic microscopy.

Unveiling mechanisms driving specification, recruitment and regeneration of melanophores is key in understanding melanin-related disorders. This study reports on the applicability of a hybrid focus optoacoustic microscope (HFOAM) for volumetric tracking of migratory melanophores in developing zebrafish. The excellent contrast from highly-absorbing melanin provided by the method is shown to be ideal for label-free dynamic visualization of melanophores in their unperturbed environment. We established safe laser energy levels that enable high-contrast longitudinal tracking of the cells over an extended period of developmental time without causing cell toxicity or pigment bleaching. Owing to its hybrid optical and acoustic resolution, the new imaging technique can be seamlessly applied for noninvasive studies of both optically-transparent larval as well as adult stages of the zebrafish model organism, which is not possible using other optical microscopy methods.

Wavelength-dependent optoacoustic imaging probes for NMDA receptor visualisation.

The cellular localisation and binding specificity of two NMDAR-targeted near-IR imaging probes has been examined by microscopy, followed by exemplification of MSOT to monitor simulated glutamate bursts in cellulo and a preliminary study in mice observing the signal in the brain.