Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics.

Tissues are complex environments where different cell types are in constant interaction with each other and with non-cellular components. Preserving the spatial context during proteomics analyses of tissue samples has become an important objective for different applications, one of the most important being the investigation of the tumor microenvironment. Here, we describe a multiplexed protein biomarker detection method on the COMET instrument, coined sequential ImmunoFluorescence (seqIF). The fully automated method uses successive applications of antibody incubation and elution, and in-situ imaging enabled by an integrated microscope and a microfluidic chip that provides optimized optical access to the sample. We show seqIF data on different sample types such as tumor and healthy tissue, including 40-plex on a single tissue section that is obtained in less than 24 h, using off-the-shelf antibodies. We also present extensive characterization of the developed method, including elution efficiency, epitope stability, repeatability and reproducibility, signal uniformity, and dynamic range, in addition to marker and panel optimization strategies. The streamlined workflow using off-the-shelf antibodies, data quality enabling downstream analysis, and ease of reaching hyperplex levels make seqIF suitable for immune-oncology research and other disciplines requiring spatial analysis, paving the way for its adoption in clinical settings.

Radical-free hyperpolarized MRI using endogenously occurring pyruvate analogues and UV-induced nonpersistent radicals.

It was recently demonstrated that nonpersistent radicals can be generated in frozen solutions of metabolites such as pyruvate by irradiation with UV light, enabling radical-free dissolution dynamic nuclear polarization. Although pyruvate is endogenous, the presence of pyruvate may interfere with metabolic processes or the detection of pyruvate as a metabolic product, making it potentially unsuitable as a polarizing agent. Therefore, the aim of the current study was to characterize solutions containing endogenously occurring alternatives to pyruvate as UV-induced nonpersistent radical precursors for in vivo hyperpolarized MRI. The metabolites alpha-ketovalerate (αkV) and alpha-ketobutyrate (αkB) are analogues of pyruvate and were chosen as potential radical precursors. Sample formulations containing αkV and αkB were studied with UV-visible spectroscopy, irradiated with UV light, and their nonpersistent radical yields were quantified with electron spin resonance and compared with pyruvate. The addition of 13 C-labeled substrates to the sample matrix altered the radical yield of the precursors. Using αkB increased the 13 C-labeled glucose liquid-state polarization to 16.3% ± 1.3% compared with 13.3% ± 1.5% obtained with pyruvate, and 8.9% ± 2.1% with αkV. For [1-13 C]butyric acid, polarization levels of 12.1% ± 1.1% for αkV, 12.9% ± 1.7% for αkB, 1.5% ± 0.2% for OX063 and 18.7% ± 0.7% for Finland trityl, were achieved. Hyperpolarized [1-13 C]butyrate metabolism in the heart revealed label incorporation into [1-13 C]acetylcarnitine, [1-13 C]acetoacetate, [1-13 C]butyrylcarnitine, [5-13 C]glutamate and [5-13 C]citrate. This study demonstrates the potential of αkV and αkB as endogenous polarizing agents for in vivo radical-free hyperpolarized MRI. UV-induced, nonpersistent radicals generated in endogenous metabolites enable high polarization without requiring radical filtration, thus simplifying the quality-control tests in clinical applications.

A robust broadband fat-suppressing phaser T2 -preparation module for cardiac magnetic resonance imaging at 3T.

PURPOSE: Designing a new T2 -preparation (T2 -Prep) module to simultaneously provide robust fat suppression and efficient T2 preparation without requiring an additional fat-suppression module for T2 -weighted imaging at 3T. METHODS: The tip-down radiofrequency (RF) pulse of an adiabatic T2 -Prep module was replaced by a custom-designed RF-excitation pulse that induces a phase difference between water and fat, resulting in a simultaneous T2 preparation of water signals and the suppression of fat signals at the end of the module (a phaser adiabatic T2 -Prep). Numerical simulations and in vitro and in vivo electrocardiogram (ECG)-triggered navigator-gated acquisitions of the human heart were performed. Blood, myocardium, and fat signal-to-noise ratios and right coronary artery vessel sharpness were compared against previously published adiabatic T2 -Prep approaches. RESULTS: Numerical simulations predicted an increased fat-suppression bandwidth and decreased sensitivity to transmit magnetic field inhomogeneities using the proposed approach while preserving the water T2 -Prep capabilities. This was confirmed by the tissue signals acquired in the phantom and the in vivo images, which show similar blood and myocardium signal-to-noise ratio, contrast-to-noise ratio, and significantly reduced fat signal-to-noise ratio compared with the other methods. As a result, the right coronary artery conspicuity was significantly increased. CONCLUSION: A novel fat-suppressing T2 -Prep method was developed and implemented that showed robust fat suppression and increased vessel sharpness compared with conventional techniques while preserving its T2 -Prep capabilities.

Controlling motion artefact levels in MR images by suspending data acquisition during periods of head motion.

PURPOSE: Head movements are a major source of MRI artefacts. Prospective motion correction techniques significantly improve data quality, but strong motion artefacts may remain in the data. We introduce a framework to suspend data acquisition during periods of head motion over a predefined threshold. METHODS: Data was acquired with prospective motion correction and an external optical tracking system. A predictor of motion impact was introduced that accounts for the amplitude of the signal acquired at the time of the motion. From this predictor, a threshold was defined to trigger the suspension of data acquisition during periods of motion. The framework was tested on 5 subjects, 2 motion behaviors, and 2 head coils (20 and 64 channels). RESULTS: The best improvements in data quality were obtained for a threshold value of 0, equivalent to suspending the acquisition based on head speed alone, at the cost of a long prolongation of scan time. For threshold values ∼3.5e-4 , image quality was largely preserved, and prolongation of scan time was minimal. Artefacts occasionally remained with the 64-channel head coil for all threshold values, seemingly due to head movement in the sharp sensitivity profile of this coil. CONCLUSION: The proposed suspension strategy is more efficient than relying on head speed alone. The threshold for suspension of data acquisition governs the tradeoff between image degradation due to motion and prolonged scan time, and can be tuned by the user according to the desired image quality and participant's tolerability.