Accelerated Screening of Protein-Ligand Interactions via Parallel T2-Weighted 19F-MRI.

In drug discovery, ligands are sought that modulate the (mal-)function of medicinally relevant target proteins. In order to develop new drugs, typically a multitude of potential ligands are initially screened for binding and subsequently characterized for their affinity. Nuclear magnetic resonance (NMR) is a well-established and highly sensitive technology for characterizing such interactions. However, it has limited throughput, because only one sample can be measured at a time. In contrast, magnetic resonance imaging (MRI) is inherently parallel and MR parameters can conveniently be encoded in its images, potentially offering increased sample throughput. We explore this application using a custom-built 9-fold sample holder and a 19F-MRI coil. With this setup, we show that ligand binding can be detected by T2-weighted 19F-MRI using 4-(trifluoromethyl)benzamidine (TFBA) and trypsin as the reporter ligand and target protein, respectively. Furthermore, we demonstrate that the affinity of nonfluorinated ligands can be determined in a competition format by monitoring the dose-dependent displacement of TFBA. By comparing 19F-T2-weighted MR images of TFBA in the presence of different benzamidine (BA) concentrations-all recorded in parallel-the affinity of BA could be derived. Therefore, this approach promises parallel characterization of protein-ligand interactions and increased throughput of biochemical assays, with potential for increased sensitivity when combined with hyperpolarization techniques.

Lenz Lenses in a Cryoprobe: Boosting NMR Sensitivity Toward Environmental Monitoring of Mass-Limited Samples.

Nuclear magnetic resonance (NMR) spectroscopy is commonly employed in a wide range of metabolomic research. Unfortunately, due to its relatively low sensitivity, smaller samples become challenging to study by NMR. Cryoprobes can be used to increase sensitivity by cooling the coil and preamplifier, offering sensitivity improvements of ∼3 to 4x. Alternatively, microcoils can be used to increase mass sensitivity by improving sample filling and proximity, along with decreased electrical resistance. Unfortunately, combining the two approaches is not just technically challenging, but as the coil decreases, so does its thermal fingerprint, reducing the advantage of cryogenic cooling. Here, an alternative solution is proposed in the form of a Lenz lens inside a cryoprobe. Rather than replacing the detection coil, Lenz lenses allow the B1 field from a larger coil to be refocused onto a much smaller sample area. In turn, the stronger B1 field at the sample provides strong coupling to the cryocoil, improving the signal. By combining a 530 I.D. Lenz lens with a cryoprobe, sensitivity was further improved by 2.8x and 3.5x for 1H and 13C, respectively, over the cryoprobe alone for small samples. Additionally, the broadband nature of the Lenz lenses allowed multiple nuclei to be studied and heteronuclear two-dimensional (2D) NMR approaches to be employed. The sensitivity improvements and 2D capabilities are demonstrated on 430 nL of hemolymph and eight eggs (∼350 µm O.D.) from the model organismDaphnia magna. In summary, combining Lenz lenses with cryoprobes offers a relatively simple approach to boost sensitivity for tiny samples while retaining cryoprobe advantages.

Topologically optimized concentric-nanoring metalens with 1 mm diameter, 0.8†NA and 600†nm imaging resolution in the visible.

Metalenses can achieve diffraction-limited focusing via localized phase modification of the incoming light beam. However, the current metalenses face to the restrictions on simultaneously achieving large diameter, large numerical aperture, broad working bandwidth and the structure manufacturability. Herein, we present a kind of metalenses composed of concentric nanorings that can address these restrictions using topology optimization approach. Compared to existing inverse design approaches, the computational cost of our optimization method is greatly reduced for large-size metalenses. With its design flexibility, the achieved metalens can work in the whole visible range with millimeter size and a numerical aperture of 0.8 without involving high-aspect ratio structures and large refractive index materials. Electron-beam resist PMMA with a low refractive index is directly used as the material of the metalens, enabling a much more simplified manufacturing process. Experimental results show that the imaging performance of the fabricated metalens has a resolution better than 600 nm corresponding to the measured FWHM of 745 nm.

The Steady-State ALTADENA RASER Generates Continuous NMR Signals.

The front cover artwork is provided by Dr. Lehmkuhl's group at the Karlsruhe Institute of Technology. The image shows continuous NMR signals complemented by a simulated bifurcation diagram of a nonlinear RASER system. Read the full text of the Research Article at 10.1002/cphc.202300204.

The Steady-State ALTADENA RASER Generates Continuous NMR Signals.

A RASER (Radio Amplification by Stimulated Emission of Radiation) facilitates the study of nonlinear phenomena, as well as the determination of NMR parameters with high precision. To achieve maximum sensitivity in the desired operating mode, it is crucial to control the RASER over long periods of time. So far, this was only possible at ultra-low magnetic fields. Here, we introduce a way to control the operating regime of a RASER at a magnetic field of 1.45 T. We employ a continuous-flow RASER, pumped by PHIP (ParaHydrogen Induced Polarization). The hydrogenation of vinyl acetate (VA) with parahydrogen provides the required negative polarization on the methyl group of the product ethyl acetate (EA). The protons within the methyl group, separated by a 7 Hz J-coupling, are RASER active. This system demonstrates five RASER phenomena: inequivalent and equivalent amplitudes in the "normal NMR mode", period doublings, frequency combs, and chaos. The experiments match with simulations based on a theoretical model of two nonlinear-coupled RASER modes. We predict the RASER regime at set conditions and visualize the prediction in a bifurcation diagram.

Highly Fluorinated Peptide Probes with Enhanced In Vivo Stability for 19 F-MRI.

A labeling strategy for in vivo 19 F-MRI (magnetic resonance imaging) based on highly fluorinated, short hydrophilic peptide probes, is developed. As dual-purpose probes, they are functionalized further by a fluorophore and an alkyne moiety for bioconjugation. High fluorination is achieved by three perfluoro-tert-butyl groups, introduced into asparagine analogues by chemically stable amide bond linkages. d-amino acids and ß-alanine in the sequences endow the peptide probes with low cytotoxicity and high serum stability. This design also yielded unstructured peptides, rendering all 27 19 F substitutions chemically equivalent, giving rise to a single 19 F-NMR resonance with <10 Hz linewidth. The resulting performance in 19 F-MRI is demonstrated for six different peptide probes. Using fluorescence microscopy, these probes are found to exhibit high stability and long circulation times in living zebrafish embryos. Furthermore, the probes can be conjugated to bovine serum albumin with only amoderate increase in 19 F-NMR linewidth to ≈30 Hz. Overall, these peptide probes are hence suitable for in vivo 19 F-MRI applications.

Analysis of U87 glioma cells by dielectrophoresis.

Glioblastoma multiforme is the most aggressive and invasive brain cancer consisting of genetically and phenotypically altering glial cells. It has massive heterogeneity due to its highly complex and dynamic microenvironment. Here, electrophysiological properties of U87 human glioma cell line were measured based on a dielectrophoresis phenomenon to quantify the population heterogeneity of glioma cells. Dielectrophoretic forces were generated using a gold-microelectrode array within a microfluidic channel when 3 Vpp and 100, 200, 300, 400, 500 kHz, 1, 2, 5, and 10 MHz frequencies were applied. We analyzed the dielectrophoretic behavior of 500 glioma cells, and revealed that the crossover frequency of glioma cells was around 140 kHz. A quantifying dielectrophoretic movement of the glioma cells exhibited three distinct glioma subpopulations: 50% of the glioma cells experienced strong, 30% of the cells were spread in the microchannel by moderate, and the rest of the cells experienced very weak positive dielectrophoretic forces. Our results demonstrated the dielectrophoretic spectra of U87 glioma cell line. Dielectrophoretic responses of glioma cells linked population heterogeneity to membrane properties of glioma cells rather than their size distribution in the population.

Simulating multilevel diffractive optical elements on a spatial light modulator.

Multilevel diffractive optical elements (DOEs) offer a solution to approximate complex diffractive phase profiles in a stepwise manner. However, while much attention has focused on efficiency, the impact on modal content in the context of structured light has, to our best knowledge, remained unexplored. Here, we outline a simple theory that accounts for efficiency and modal purity in arbitrary structured light produced by multilevel DOEs. We make use of a phase-only spatial light modulator as a "testbed" to experimentally implement various multileveled diffractive profiles, including orbital angular momentum beams, Bessel beams, and Airy beams, outlining the subsequent efficiency and purity both theoretically and experimentally, confirming that a low number of multilevel steps can produce modes of high fidelity. Our work will be useful to those wishing to digitally evaluate modal effects from DOEs prior to physical fabrication.

Real-Time NMR Monitoring of Spatially Segregated Enzymatic Reactions in Multilayered Hydrogel Assemblies*.

Compartmentalized chemical reactions at the microscale are important in biotechnology, yet monitoring the molecular content at these small scales is challenging. To address this challenge, we integrate a compact, reconfigurable reaction cell featuring electrochemical functionality with high-resolution NMR spectroscopy. We demonstrate the operation of this system by monitoring the activity of enzymes immobilized in chemically distinct layers within a multi-layered chitosan hydrogel assembly. As a benchmark, we observed the parallel activities of urease (Urs), catalase (Cat), and glucose oxidase (GOx) by monitoring reagent and product concentrations in real-time. Simultaneous monitoring of an independent enzymatic process (Urs) together with a cooperative process (GOx + Cat) was achieved, with chemical conversion modulation of the GOx + Cat process demonstrated by varying the order in which the hydrogel was assembled.

Motion prediction enables simulated MR-imaging of freely moving model organisms.

Magnetic resonance tomography typically applies the Fourier transform to k-space signals repeatedly acquired from a frequency encoded spatial region of interest, therefore requiring a stationary object during scanning. Any movement of the object results in phase errors in the recorded signal, leading to deformed images, phantoms, and artifacts, since the encoded information does not originate from the intended region of the object. However, if the type and magnitude of movement is known instantaneously, the scanner or the reconstruction algorithm could be adjusted to compensate for the movement, directly allowing high quality imaging with non-stationary objects. This would be an enormous boon to studies that tie cell metabolomics to spontaneous organism behaviour, eliminating the stress otherwise necessitated by restraining measures such as anesthesia or clamping. In the present theoretical study, we use a phantom of the animal model C. elegans to examine the feasibility to automatically predict its movement and position, and to evaluate the impact of movement prediction, within a sufficiently long time horizon, on image reconstruction. For this purpose, we use automated image processing to annotate body parts in freely moving C. elegans, and predict their path of movement. We further introduce an MRI simulation platform based on bright field videos of the moving worm, combined with a stack of high resolution transmission electron microscope (TEM) slice images as virtual high resolution phantoms. A phantom provides an indication of the spatial distribution of signal-generating nuclei on a particular imaging slice. We show that adjustment of the scanning to the predicted movements strongly reduces distortions in the resulting image, opening the door for implementation in a high-resolution NMR scanner.

Automatic Adaptive Gain for Magnetic Resonance Sensitivity Enhancement.

The decaying nature of magnetic resonance (MR) signals results in a decreasing signal-to-quantization noise ratio (SQNR) over the acquisition time. Here we describe a method to enhance the SQNR, and thus the overall signal-to-noise ratio (SNR), by dynamically adapting the gain of the receiver before analog-to-digital conversion (ADC). This is in contrast to a standard experiment in which the gain is fixed for a single data acquisition and is thus adjusted only for the first points of the signal. The gain adjustment in our method is done automatically in a closed loop fashion by using the envelope of the MR signal as the control signal. Moreover, the method incorporates a robust mechanism that runs along with signal acquisition to monitor the gain modulation, enabling precise recovery of the signals. The automatic adaptive gain (AGAIN) method requires minimal additional hardware and is thus general and can be implemented in the signal path of any commercial spectrometer system. We demonstrate an SNR enhancement factor of 2.64 when applied to a custom spectrometer, while a factor of 1.4 was observed when applied to a commercial spectrometer.

Microfluidic Chips for Life Sciences-A Comparison of Low Entry Manufacturing Technologies.

Microfluidic water-in-oil droplets are a versatile tool for biological and biochemical applications due to the advantages of extremely small monodisperse reaction vessels in the pL-nL range. A key factor for the successful dissemination of this technology to life science laboratory users is the ability to produce microfluidic droplet generators and related accessories by low-entry barrier methods, which enable rapid prototyping and manufacturing of devices with low instrument and material costs. The direct, experimental side-by-side comparison of three commonly used additive manufacturing (AM) methods, namely fused deposition modeling (FDM), inkjet printing (InkJ), and stereolithography (SLA), is reported. As a benchmark, micromilling (MM) is used as an established method. To demonstrate which of these methods can be easily applied by the non-expert to realize applications in topical fields of biochemistry and microbiology, the methods are evaluated with regard to their limits for the minimum structure resolution in all three spatial directions. The suitability of functional SLA and MM chips to replace classic SU-8 prototypes is demonstrated on the basis of representative application cases.

Spatial scanning hyperspectral imaging combining a rotating slit with a Dove prism.

We present a new concept for spatial scanning hyperspectral imaging. Spatial scanning is one of the main methods used for hyperspectral data acquisition and can provide high spectral resolution over a wide spectral range. However, conventional techniques, such as the whiskbroom and the pushbroom techniques, suffer from the need for relative motion between the target and the imaging system, which increases the complexity on the hardware side and limits the application possibilities. Our new approach combines a rotating slit and a co-rotating Dove prism. The rotating slit scans the target image by selecting one line from the image at each angular position of the slit. The rotating Dove prism is used to synchronously re-align the transmitted light from the selected image line with respect to the transmission grating to allow the projection of the diffracted light over the same range of pixel columns of the image sensor to facilitate data acquisition and extraction of spectral information. The new approach enables the spatial scanning of the target image without the need for relative linear motion or the use of additional external equipment and therefore opens the door for more application scenarios.

Inductively coupled magic angle spinning microresonators benchmarked for high-resolution single embryo metabolomic profiling.

The magic angle coil spinning (MACS) technique has been introduced as a very promising extension for solid state NMR detection, demonstrating sensitivity enhancements by a factor of 14 from the very first time it has been reported. The main beneficiary of this technique is the scientific community dealing with mass- and volume-limited, rare, or expensive samples. However, more than a decade after the first report on MACS, there is a very limited number of groups who have continued to develop the technique, let alone it being widely adopted by practitioners. This might be due to several drawbacks associated with the MACS technology until now, including spectral linewidth, heating due to eddy currents, and imprecise manufacturing. Here, we report a device overcoming all these remaining issues, therefore achieving: (1) spectral resolution of approx 0.01 ppm and normalized limit of detection of approx. 13 nmol s0.5 calculated using the anomeric proton of sucrose at 3 kHz MAS frequency; (2) limited temperature increase inside the MACS insert of only 5 °C at 5 kHz MAS frequency in an 11.74 T magnetic field, rendering MACS suitable to study live biological samples. The wafer-scale fabrication process yields MACS inserts with reproducible properties, readily available to be used on a large scale in bio-chemistry labs. To illustrate the potential of these devices for metabolomic studies, we further report on: (3) ultra-fine 1H-1H and 13C-13C J-couplings resolved within 10 min for a 340 mM uniformly 13C-labeled glucose sample; and (4) single zebrafish embryo measurements through 1H-1H COSY within 4.5 h, opening the gate for the single embryo NMR studies.

On the application of balanced steady-state free precession to MR microscopy.

OBJECTIVE: The applicability of the balanced steady-state free precession (bSSFP) sequence to the field of MR microscopy was investigated, since the potentially high SNR makes bSSFP attractive. However, particularly at ultra-high magnetic fields, a number of constraints emerge: the frequency sensitivity of the bSSFP signal, the duty cycle of the imaging gradients, and the intrinsic diffusion attenuation of the steady state due to the imaging gradients. MATERIALS AND METHODS: Optimization of the bSSFP sequence was performed on three imaging systems (7 T and 9.4 T) suited for MR microscopy. Since biological samples are often imaged in the very proximity of materials from sample containers/holder or devices such as electrodes, several microscopy phantoms representing such circumstances were fabricated and examined with 3D bSSFP. RESULTS: Artifact-free microscopic bSSFP images could be obtained with voxel sizes down to 16 µm × 16 µm × 78 µm and with an SNR gain of 25% over standard gradient echo images. CONCLUSION: With appropriate choice of phantom materials, optimization of the flip angle to the diffusion-attenuated steady state and protocols considering duty-cycle limitations, bSSFP can be a valuable tool in MR microscopy.

Correction: Design and Simulation of a Wireless SAW-Pirani Sensor with Extended Range and Sensitivity.

The authors wish to make the following erratum to Reference [...].

Design and Simulation of a Wireless SAW-Pirani Sensor with Extended Range and Sensitivity.

Pressure is a critical parameter for a large number of industrial processes. The vacuum industry relies on accurate pressure measurement and control. A new compact wireless vacuum sensor was designed and simulated and is presented in this publication. The sensor combines the Pirani principle and Surface Acoustic Waves, and it extends the vacuum sensed range to between 10-4 Pa and 105 Pa all along a complete wireless operation. A thermal analysis was performed based on gas kinetic theory, aiming to optimize the thermal conductivity and the Knudsen regime of the device. Theoretical analysis and simulation allowed designing the structure of the sensor and its dimensions to ensure the highest sensitivity through the whole sensing range and to build a model that simulates the behavior of the sensor under vacuum. A completely new design and a model simulating the behavior of the sensor from high vacuum to atmospheric pressure were established.

One-second MRI of a three-dimensional vocal tract to measure dynamic articulator modifications.

PURPOSE: To enable three-dimensional (3D) vocal tract imaging of dynamic singing or speech tasks at voxel sizes of 1.6 × 1.6 × 1.3 mm3 at 1.3 s per image. MATERIALS AND METHODS: A Stack-of-Stars method was implemented and enhanced to allow for fast and efficient k-space sampling of the box-shaped vocal tract using a 3 Tesla MRI system. Images were reconstructed using an off-line image reconstruction using compressed sensing theory, leading to the abovementioned spatial and temporal resolutions. To validate spatial resolution, a phantom with holes of defined sizes was measured. The applicability of the imaging method was validated in an eight-subject study of amateur singers that were required to sustain phonation at a constant pitch, past their comfortable expiratory level. A segmentation of the vocal tract over all phonation time steps was done for one subject. Anatomical distances (larynx position and pharynx width) were calculated and compared for all subjects. RESULTS: Analysis of the phantom study revealed that the imaging method could provide at least 1.6 mm isotropic resolution. Visual inspection of the segmented vocal tract during phonation showed modifications of the lips, tongue, and larynx position in all three dimensions. The mean larynx position per subject amounted to 52-85 mm, deviating up to 5% over phonation time. Parameter pharynx width was 32-181 mm2 on average per subject, deviating up to 16% over phonation time. Visual inspection of the parameter course revealed no common compensation strategy for long sustained phonation. CONCLUSION: The results of both phantom and in vivo measurements show the applicability of the fast 3D imaging method for voice research and indicate that modifications in all three dimensions can be observed and quantified. LEVEL OF EVIDENCE: 2 Technical Efficacy: Stage 1 J. MAGN. RESON. IMAGING 2017;46:94-101.

A comparison of Lenz lenses and LC resonators for NMR signal enhancement.

High signal-to-noise ratio (SNR) of the NMR signal has always been a key target that drives massive research effort in many fields. Among several parameters, a high filling factor of the MR coil has proven to boost the SNR. In case of small-volume samples, a high filling factor and thus a high SNR can be achieved through miniaturizing the MR coil. However, under certain circumstances, this can be impractical. In this paper, we present an extensive theoretical and experimental investigation of the inductively coupled LC resonator and the magnetic Lenz lens as two candidate approaches that can enhance the SNR in such circumstances. The results demonstrate that the narrow-band LC resonator is superior in terms of SNR, while the non-tuned nature of the Lenz lens makes it preferable in broadband applications.

Dissipative particle dynamics of diffusion-NMR requires high Schmidt-numbers.

We present an efficient mesoscale model to simulate the diffusion measurement with nuclear magnetic resonance (NMR). On the level of mesoscopic thermal motion of fluid particles, we couple the Bloch equations with dissipative particle dynamics (DPD). Thereby we establish a physically consistent scaling relation between the diffusion constant measured for DPD-particles and the diffusion constant of a real fluid. The latter is based on a splitting into a centre-of-mass contribution represented by DPD, and an internal contribution which is not resolved in the DPD-level of description. As a consequence, simulating the centre-of-mass contribution with DPD requires high Schmidt numbers. After a verification for fundamental pulse sequences, we apply the NMR-DPD method to NMR diffusion measurements of anisotropic fluids, and of fluids restricted by walls of microfluidic channels. For the latter, the free diffusion and the localisation regime are considered.