Martin Kunth

Cell Tracking with Caged Xenon: Using Cryptophanes as MRI Reporters upon Cellular Internalization

Caged xenon has great potential in overcoming sensitivity limitations for solution-state NMR detection of dilute molecules. However, no application of such a system as a magnetic resonance imaging (MRI) contrast agent has yet been performed with live cells. We demonstrate MRI localization of cells labeled with caged xenon in a packed-bed bioreactor working under perfusion with hyperpolarized-xenon-saturated medium. Xenon hosts enable NMR/MRI experiments with switchable contrast and selectivity for cell-associated versus unbound cages. We present MR images with 10(3) -fold sensitivity enhancement for cell-internalized, dual-mode (fluorescence/MRI) xenon hosts at low micromolar concentrations. Our results illustrate the capability of functionalized xenon to act as a highly sensitive cell tracer for MRI detection even without signal averaging. The method will bridge the challenging gap for translation to in vivo studies for the optimization of targeted biosensors and their multiplexing applications.

Continuous-wave saturation considerations for efficient xenon depolarization

The combination of hyperpolarized Xe with chemical exchange saturation transfer (Hyper‐CEST) is a powerful NMR technique to detect highly dilute concentrations of Xe binding sites using RF saturation pulses. Crucially, that combination of saturation pulse strength and duration that generates the maximal Hyper‐CEST effect is a priori unknown. In contrast to CEST in proton MRI, where the system reaches a steady‐state for long saturation times, Hyper‐CEST has an optimal saturation time, i.e. saturating for shorter or longer reduces the Hyper‐CEST effect. Here, we derive expressions for this optimal saturation pulse length. We also found that a pulse strength, B1, corresponding to five times the Xe exchange rate, kBA (i.e. B1 = 5 kBA/γ with the gyromagnetic ratio of 129Xe, γ), generates directly and without further optimization 96 % of the maximal Hyper‐CEST contrast while preserving spectral selectivity. As a measure that optimizes the amplitude and the width of the Hyper‐CEST response simultaneously, we found an optimal saturation pulse strength corresponding to 2 times the Xe exchange rate, i.e.  B1=2kBA/γ . When extremely low host concentration is detected, then the expression for the optimum saturation time simplifies as it approaches the longitudinal relaxation time of free Xe. Copyright

Sensitivity enhancement of (Hyper-)CEST image series by exploiting redundancies in the spectral domain

CEST has proven to be a valuable technique for the detection of hyperpolarized xenon-based functionalized contrast agents. Additional information can be encoded in the spectral dimension, allowing the simultaneous detection of multiple different biosensors. However, owing to the low concentration of dissolved xenon in biological tissue, the signal-to-noise ratio (SNR) of Hyper-CEST data is still a critical issue. In this work, we present two techniques aiming to increase SNR by exploiting the typically high redundancy in spectral CEST image series: PCA-based post-processing and sub-sampled acquisition with low-rank reconstruction. Each of them yields a significant SNR enhancement, demonstrating the feasibility of the two approaches. While the first method is directly applicable to proton CEST experiments as well, the second one is particularly beneficial when dealing with hyperpolarized nuclei, since it distributes the non-renewable initial polarization more efficiently over the sampling points. The results obtained are a further step towards the detection of xenon biosensors with spectral Hyper-CEST imaging in vivo.

Hyperpolarized Xenon for NMR and MRI Applications

Nuclear magnetic resonance (NMR) spectroscopy and imaging (MRI) suffer from intrinsic low sensitivity because even strong external magnetic fields of ~10 T generate only a small detectable net-magnetization of the sample at room temperature (1). Hence, most NMR and MRI applications rely on the detection of molecules at relative high concentration (e.g., water for imaging of biological tissue) or require excessive acquisition times. This limits our ability to exploit the very useful molecular specificity of NMR signals for many biochemical and medical applications. However, novel approaches have emerged in the past few years: Manipulation of the detected spin species prior to detection inside the NMR/MRI magnet can dramatically increase the magnetization and therefore allows detection of molecules at much lower concentration (2). Here, we present a method for polarization of a xenon gas mixture (2-5% Xe, 10% N2, He balance) in a compact setup with a ca. 16000-fold signal enhancement. Modern line-narrowed diode lasers allow efficient polarization (7) and immediate use of gas mixture even if the noble gas is not separated from the other components. The SEOP apparatus is explained and determination of the achieved spin polarization is demonstrated for performance control of the method. The hyperpolarized gas can be used for void space imaging, including gas flow imaging or diffusion studies at the interfaces with other materials (8,9). Moreover, the Xe NMR signal is extremely sensitive to its molecular environment (6). This enables the option to use it as an NMR/MRI contrast agent when dissolved in aqueous solution with functionalized molecular hosts that temporarily trap the gas (10,11). Direct detection and high-sensitivity indirect detection of such constructs is demonstrated in both spectroscopic and imaging mode.

Preparation of biogenic gas vesicle nanostructures for use as contrast agents for ultrasound and MRI

Gas vesicles (GVs) are a unique class of gas-filled protein nanostructures that are detectable at subnanomolar concentrations and whose physical properties allow them to serve as highly sensitive imaging agents for ultrasound and MRI. Here we provide a protocol for isolating GVs from native and heterologous host organisms, functionalizing these nanostructures with moieties for targeting and fluorescence, characterizing their biophysical properties and imaging them using ultrasound and MRI. GVs can be isolated from natural cyanobacterial and haloarchaeal host organisms or from Escherichia coli expressing a heterologous GV gene cluster and purified using buoyancy-assisted techniques. They can then be modified by replacing surface-bound proteins with engineered, heterologously expressed variants or through chemical conjugation, resulting in altered mechanical, surface and targeting properties. Pressurized absorbance spectroscopy is used to characterize their mechanical properties, whereas dynamic light scattering (DLS)and transmission electron microscopy (TEM) are used to determine nanoparticle size and morphology, respectively. GVs can then be imaged with ultrasound in vitro and in vivo using pulse sequences optimized for their detection versus background. They can also be imaged with hyperpolarized xenon MRI using chemical exchange saturation transfer between GV-bound and dissolved xenon—a technique currently implemented in vitro. Taking 3–8 d to prepare, these genetically encodable nanostructures enable multimodal, noninvasive biological imaging with high sensitivity and potential for molecular targeting.

Time-resolved Monitoring of Enzyme Activity with Ultrafast Hyper-CEST Spectroscopy

We propose a method to dynamically monitor the progress of an enzymatic reaction using NMR of hyperpolarized 129Xe in a host–guest system. It is based on a displacement assay originally designed for fluorescence experiments that exploits the competitive binding of the enzymatic product on the one hand and a reporter dye on the other hand to a supramolecular host. Recently, this assay has been successfully transferred to NMR, using xenon as a reporter, cucurbit[6]uril as supramolecular host, and chemical exchange saturation transfer with hyperpolarized Xe (Hyper‐CEST) as detection technique. Its advantage is that the enzyme acts on the unmodified substrate and that only the product is detected through immediate inclusion into the host. We here apply a method that drastically accelerates the acquisition of Hyper‐CEST spectra in vitro using magnetic field gradients. This allows monitoring the dynamic progress of the conversion of lysine to cadaverine with a temporal resolution of ~30 s. Moreover, the method only requires to sample the very early onset of the reaction (<0.5% of substrate conversion where the host itself is required only at μM concentrations) at comparatively low reaction rates, thus saving enzyme material and reducing NMR acquisition time. The obtained value for the specific activity agrees well with previously published results from fluorescence assays. We furthermore outline how the Hyper‐CEST results correlate with xenon T2 measurements performed during the enzymatic reaction. This suggests that ultrafast Hyper‐CEST spectroscopy can be used for dynamically monitoring enzymatic activity with NMR.

Recombinantly Expressed Gas Vesicles as Nanoscale Contrast Agents for Ultrasound and Hyperpolarized MRI

Ultrasound and hyperpolarized magnetic resonance imaging enable the visualization of biological processes in deep tissues. However, few molecular contrast agents are available to connect these modalities to specific aspects of biological function. We recently discovered that a unique class of gas-filled protein nanostructures known as gas vesicles could serve as nanoscale molecular reporters for these modalities. However, the need to produce these nanostructures via expression in specialized cultures of cyanobacteria or haloarchaea limits their broader adoption by other laboratories and hinders genetic engineering of their properties. Here, we describe recombinant expression and purification of Bacillus megaterium gas vesicles using a common laboratory strain of Escherichia coli, and characterize the physical, acoustic and magnetic resonance properties of these nanostructures. Recombinantly expressed gas vesicles produce ultrasound and hyperpolarized 129Xe MRI contrast at sub-nanomolar concentrations, thus validating a simple platform for their production and engineering.

Protein Nanostructures Produce Self-Adjusting Hyperpolarized Magnetic Resonance Imaging Contrast through Physical Gas Partitioning

Signal amplification strategies are critical for overcoming the intrinsically poor sensitivity of nuclear magnetic resonance (NMR) reporters in noninvasive molecular detection. A mechanism widely used for signal enhancement is chemical exchange saturation transfer (CEST) of nuclei between a dilute sensing pool and an abundant detection pool. However, the dependence of CEST amplification on the relative size of these spin pools confounds quantitative molecular detection with a larger detection pool typically making saturation transfer less efficient. Here we show that a recently discovered class of genetically encoded nanoscale reporters for 129Xe magnetic resonance overcomes this fundamental limitation through an elastic binding capacity for NMR-active nuclei. This approach pairs high signal amplification from hyperpolarized spins with ideal, self-adjusting saturation transfer behavior as the overall spin ensemble changes in size. These reporters are based on gas vesicles, i.e., microbe-derived, gas-filled protein nanostructures. We show that the xenon fraction that partitions into gas vesicles follows the ideal gas law, allowing the signal transfer under hyperpolarized xenon chemical exchange saturation transfer (Hyper-CEST) imaging to scale linearly with the total xenon ensemble. This conceptually distinct elastic response allows the production of quantitative signal contrast that is robust to variability in the concentration of xenon, enabling virtually unlimited improvement in absolute contrast with increased xenon delivery, and establishing a unique principle of operation for contrast agent development in emerging biochemical and in vivo applications of hyperpolarized NMR and magnetic resonance imaging.