Fluorinated macromolecular amphiphiles as prototypic molecular drones.

The advent of drones has revolutionized various aspects of our lives, and in the realm of biological systems, molecular drones hold immense promise as "magic bullets" for major diseases. Herein, we introduce a unique class of fluorinated macromolecular amphiphiles, designed in the shape of jellyfish, serving as exemplary molecular drones for fluorine-19 MRI (19F MRI) and fluorescence imaging (FLI)-guided drug delivery, status reporting, and targeted cancer therapy. Functioning akin to their mechanical counterparts, these biocompatible molecular drones autonomously assemble with hydrophobic drugs to form uniform nanoparticles, facilitating efficient drug delivery into cells. The status of drug delivery can be tracked through aggregation-induced emission (AIE) of FLI and 19F MRI. Furthermore, when loaded with a heptamethine cyanine fluorescent dye IR-780, these molecular drones enable near-infrared (NIR) FL detection of tumors and precise delivery of the photosensitizer. Similarly, when loaded with doxorubicin (DOX), they enable targeted chemotherapy with fluorescence resonance energy transfer (FRET) FL for real-time status updates, resulting in enhanced therapeutic efficacy. Compared to conventional drug delivery systems, molecular drones stand out for their simplicity, precise structure, versatility, and ability to provide instantaneous status updates. This study presents prototype molecular drones capable of executing fundamental drone functions, laying the groundwork for the development of more sophisticated molecular machines with significant biomedical implications.

Self-assembling dendrimer nanosystems for specific fluorine magnetic resonance imaging and effective theranostic treatment of tumors.

Fluorine magnetic resonance imaging (19F-MRI) is particularly promising for biomedical applications owing to the absence of fluorine in most biological systems. However, its use has been limited by the lack of safe and water-soluble imaging agents with high fluorine contents and suitable relaxation properties. We report innovative 19F-MRI agents based on supramolecular dendrimers self-assembled by an amphiphilic dendrimer composed of a hydrophobic alkyl chain and a hydrophilic dendron. Specifically, this amphiphilic dendrimer bears multiple negatively charged terminals with high fluorine content, which effectively prevented intra- and intermolecular aggregation of fluorinated entities via electrostatic repulsion. This permitted high fluorine nuclei mobility alongside good water solubility with favorable relaxation properties for use in 19F-MRI. Importantly, the self-assembling 19F-MRI agent was able to encapsulate the near-infrared fluorescence (NIRF) agent DiR and the anticancer drug paclitaxel for multimodal 19F-MRI and NIRF imaging of and theranostics for pancreatic cancer, a deadly disease for which there remains no adequate early detection method or efficacious treatment. The 19F-MRI and multimodal 19F-MRI and NIRF imaging studies on human pancreatic cancer xenografts in mice confirmed the capability of both imaging modalities to specifically image the tumors and demonstrated the efficacy of the theranostic agent in cancer treatment, largely outperforming the clinical anticancer drug paclitaxel. Consequently, these dendrimer nanosystems constitute promising 19F-MRI agents for effective cancer management. This study offers a broad avenue to the construction of 19F-MRI agents and theranostics, exploiting self-assembling supramolecular dendrimer chemistry.

Fluorinated 1,7-DO2A-Based Iron(II) Complexes as Sensitive 19F MRI Molecular Probes for Visualizing Renal Dysfunction in Living Mice.

Kidney diseases have become an important global health concern due to their high incidence, inefficient diagnosis, and poor prognosis. Devising direct methods, especially imaging means, to assess renal function is the key for better understanding the mechanisms of various kidney diseases and subsequent development of effective treatment. Herein, we developed a fluorinated ferrous chelate-based sensitive probe, 1,7-DO2A-Fe(II)-F18 (Probe 1), for 19F magnetic resonance imaging (MRI). This highly fluorinated probe (containing 18 chemically equivalent 19F atoms with a fluorine content at 35 wt %) achieves a 15-time enhancement in signal intensity compared with the fluorine-containing ligand alone due to the appropriately regulated 19F relaxation times by the ferrous ion, which significantly increases imaging sensitivity and reduces acquisition time. Owing to its high aqueous solubility, biostability, and biocompatibility, this probe could be rapidly cleared by kidneys, which provides a means for monitoring renal dysfunction via 19F MRI. With this probe, we accomplish in vivo imaging of the impaired renal dysfunction caused by various kidney diseases including acute kidney injury, unilateral ureteral obstruction, and renal fibrosis at different stages. Our study illustrates the promising potential of Probe 1 for in vivo real-time visualization of kidney dysfunction, which is beneficial for the study, diagnosis, and even stratification of different kidney diseases. Furthermore, the design strategy of our probe is inspiring for the development of more high-performance 19F MRI probes for monitoring various biological processes.

A stable, highly concentrated fluorous nanoemulsion formulation for in vivo cancer imaging via 19F-MRI.

Magnetic resonance imaging (MRI) is a routine diagnostic modality in oncology that produces excellent imaging resolution and tumor contrast without the use of ionizing radiation. However, improved contrast agents are still needed to further increase detection sensitivity and avoid toxicity/allergic reactions associated with paramagnetic metal contrast agents, which may be seen in a small percentage of the human population. Fluorine-19 (19F)-MRI is at the forefront of the developing MRI methodologies due to near-zero background signal, high natural abundance of 100%, and unambiguous signal specificity. In this study, we have developed a colloidal nanoemulsion (NE) formulation that can encapsulate high volumes of the fluorous MRI tracer, perfluoro-[15-crown-5]-ether (PFCE) (35% v/v). These nanoparticles exhibit long-term (at least 100 days) stability and high PFCE loading capacity in formulation with our semifluorinated triblock copolymer, M2F8H18. With sizes of approximately 200 nm, these NEs enable in vivo delivery and passive targeting to tumors. Our diagnostic formulation, M2F8H18/PFCE NE, yielded in vivo 19F-MR images with a high signal-to-noise ratio up to 100 in a tumor-bearing mouse model at clinically relevant scan times. M2F8H18/PFCE NE circulated stably in the vasculature, accumulated in high concentration of an estimated 4-9 × 1017 19F spins/voxel at the tumor site, and cleared from most organs over the span of 2 weeks. Uptake by the mononuclear phagocyte system to the liver and spleen was also observed, most likely due to particle size. These promising results suggest that M2F8H18/PFCE NE is a favorable 19F-MR diagnostic tracer for further development in oncological studies and potential clinical translation.

Stimuli-Responsive Polymers for Advanced 19F Magnetic Resonance Imaging: From Chemical Design to Biomedical Applications.

Fluorine magnetic resonance imaging (19F MRI) is a rapidly evolving research area with a high potential to advance the field of clinical diagnostics. In this review, we provide an overview of the recent progress in the field of fluorinated stimuli-responsive polymers applied as 19F MRI tracers. These polymers respond to internal or external stimuli (e.g., temperature, pH, oxidative stress, and specific molecules) by altering their physicochemical properties, such as self-assembly, drug release, and polymer degradation. Incorporating noninvasive 19F labels enables us to track the biodistribution of such polymers. Furthermore, by triggering polymer transformation, we can induce changes in 19F MRI signals, including attenuation, amplification, and chemical shift changes, to monitor alterations in the environment of the tracer. Ultimately, this review highlights the emerging potential of stimuli-responsive fluoropolymer 19F MRI tracers in the current context of polymer diagnostics research.

Hyperbranched Polymeric 19F MRI Contrast Agents with Long T2 Relaxation Time Based on ß-Cyclodextrin and Phosphorycholine.

19F magnetic resonance imaging (19F MRI) is gaining attention as an emerging diagnostic technology. Effective 19F MRI contrast agents (CAs) for in vivo applications require a long transverse (or spin-spin) relaxation time (T2), short longitudinal (or spin-lattice) relaxation time (T1), high fluorine content, and excellent biocompatibility. Here, we present a novel hyperbranched polymeric 19F MRI CA based on ß-cyclodextrin and phosphorylcholine. The influence of the branching degree and fluorine content on T2 was thoroughly investigated. Results demonstrated a maximum fluorine content of 11.85% and a T2 of 612 ms. This hyperbranched polymeric 19F MRI CA exhibited both great biocompatibility against cells and organs of mice and high-performance imaging capabilities both in vitro and in vivo. The research provides positive insights into the synthesis strategies, topological design, and selection of fluorine tags for 19F MRI CAs.

Synthesis of fluorinated curcumin derivatives for detecting amyloid plaques by 19F-MRI.

The most prominent pathophysiological hallmark of Alzheimer's disease is the aggregation of amyloid-ß (Aß) peptides into senile plaques. Curcumin and its derivatives exhibit a high affinity for binding to Aß fibrils, effectively inhibiting their growth. This property holds promise for both therapeutic applications and diagnostic molecular imaging. In this study, curcumin was functionalized with perfluoro-tert-butyl groups to create candidate molecular probes specifically targeted to Aß fibrils for use in 19F-magnetic resonance imaging. Two types of fluorinated derivatives were considered: mono-substituted (containing nine fluorine atoms per molecule) and disubstituted (containing eighteen fluorine atoms). The linker connecting the perfluoro moiety with the curcumin scaffold was evaluated for its impact on binding affinity and water solubility. All mono-substituted compounds and one disubstituted compound exhibited a binding affinity toward Aß fibrils on the same order of magnitude as reference curcumin. The insertion of a charged carboxylate group into the linker enhanced the water solubility of the probes. Compound Curc-Glu-F9 (with one L-glutamyl moiety and a perfluoro-tert-butyl group), showed the best properties in terms of binding affinity towards Aß fibrils, water solubility, and intensity of the 19F-NMR signal in the Aß oligomer bound form.

Quantifying Micromolar Crystallinity in Pharmaceutical Materials Utilizing 19F Solid-State NMR.

Quantifying low-level components in solid-state analysis presents a significant challenge for most thermal, diffractometric, vibrational, and spectroscopic techniques. In pharmaceutical analysis, identifying and quantifying the physical form of the drug substance in solid dosages is a critical task to ensure the quality of drug products. For example, recrystallization of active pharmaceutical ingredients in amorphous solid dispersions can compromise the stability and bioavailability of drug products. Herein, we have developed and demonstrated fluorine-19 solid-state nuclear magnetic resonance (19F ssNMR) methods and pushed the boundary to quantify minor crystalline contents in amorphous pharmaceuticals. Calibration curves suggest that 19F direct polarization and 1H-19F cross-polarization ssNMR can readily quantify 0.1% w/w crystalline compound I, a commercial fluorinated drug molecule developed by Merck & Co., Inc., Rahway, NJ, U.S.A., in its amorphous formulation. 1H-19F multiple cross-polarization (MultiCP) has been implemented, for the first time, and compared with conventional cross-polarization methods. Most importantly, a relaxation-filtered 19F ssNMR method was utilized to unambiguously identify and quantify as low as 0.04% w/w crystalline components, that is, 6 µmol in a 100 mg tablet at 25% drug loading, by suppressing the signal from the amorphous counterpart. Such a low level of detection offers high confidence and sensitivity to quantify trace amounts of phase change in pharmaceutical amorphous materials in the solid state, which can facilitate formulation development as well as quality control.

(R)-α-Trifluoromethylalanine as a 19 F NMR Probe for the Monitoring of Protease Digestion of Peptides.

Fluorinated non-natural amino acids are useful tools for improving the bioavailability of peptides but can also serve as fluorinated probes in 19 F NMR-based enzymatic assays. We report herein that the use of the non-natural α-quaternarized (R)-α-trifluoromethylalanine ((R)-α-TfmAla) provides convenient and accurate monitoring of trypsin proteolytic activity and increases resistance towards pepsin degradation.

Enhanced detection of paramagnetic fluorine-19 magnetic resonance imaging agents using zero echo time sequence and compressed sensing.

Fluorine-19 (19 F) magnetic resonance imaging (MRI) is an emerging technique offering specific detection of labeled cells in vivo. Lengthy acquisition times and modest signal-to-noise ratio (SNR) makes three-dimensional spin-density-weighted 19 F imaging challenging. Recent advances in tracer paramagnetic metallo-perfluorocarbon (MPFC) nanoemulsion probes have shown multifold SNR improvements due to an accelerated 19 F T1 relaxation rate and a commensurate gain in imaging speed and averages. However, 19 F T2 -reduction and increased linewidth limit the amount of metal additive in MPFC probes, thus constraining the ultimate SNR. To overcome these barriers, we describe a compressed sampling (CS) scheme, implemented using a "zero" echo time (ZTE) sequence, with data reconstructed via a sparsity-promoting algorithm. Our CS-ZTE scheme acquires k-space data using an undersampled spherical radial pattern and signal averaging. Image reconstruction employs off-the-shelf sparse solvers to solve a joint total variation and l1 -norm regularized least square problem. To evaluate CS-ZTE, we performed simulations and acquired 19 F MRI data at 11.7 T in phantoms and mice receiving MPFC-labeled dendritic cells. For MPFC-labeled cells in vivo, we show SNR gains of ~6.3 × with 8-fold undersampling. We show that this enhancement is due to three mechanisms including undersampling and commensurate increase in signal averaging in a fixed scan time, denoising attributes from the CS algorithm, and paramagnetic reduction of T1 . Importantly, 19 F image intensity analyses yield accurate estimates of absolute quantification of 19 F spins. Overall, the CS-ZTE method using MPFC probes achieves ultrafast imaging, a substantial boost in detection sensitivity, accurate 19 F spin quantification, and minimal image artifacts.

Site-Specific Labeling and 19F NMR Provide Direct Evidence for Dynamic Behavior of the Anthrax Toxin Pore ϕ-Clamp Structure.

The anthrax toxin protective antigen (PA), the membrane binding and pore-forming component of the anthrax toxin, was studied using 19F NMR. We site-specifically labeled PA with p-fluorophenylalanine (pF-Phe) at Phe427, a critically important residue that comprises the ϕ-clamp that is required for translocation of edema factor (EF) and lethal factor (LF) into the host cell cytosol. We utilized 19F NMR to follow low-pH-induced structural changes in the prepore, alone and bound to the N-terminal PA binding domain of LF, LFN. Our studies indicate that pF-Phe427 is dynamic in the prepore state and then becomes more dynamic in the transition to the pore. An increase in dynamic behavior at the ϕ-clamp may provide the necessary room for movement needed in translocating EF and LF into the cell cytosol.

Protein-observed 19F NMR of LecA from Pseudomonas aeruginosa.

The carbohydrate-binding protein LecA (PA-IL) from Pseudomonas aeruginosa plays an important role in the formation of biofilms in chronic infections. Development of inhibitors to disrupt LecA-mediated biofilms is desired but it is limited to carbohydrate-based ligands. Moreover, discovery of drug-like ligands for LecA is challenging because of its weak affinities. Therefore, we established a protein-observed 19F (PrOF) nuclear magnetic resonance (NMR) to probe ligand binding to LecA. LecA was labeled with 5-fluoroindole to incorporate 5-fluorotryptophanes and the resonances were assigned by site-directed mutagenesis. This incorporation did not disrupt LecA preference for natural ligands, Ca2+ and d-galactose. Following NMR perturbation of W42, which is located in the carbohydrate-binding region of LecA, allowed to monitor binding of low-affinity ligands such as N-acetyl d-galactosamine (d-GalNAc, Kd = 780 ± 97 µM). Moreover, PrOF NMR titration with glycomimetic of LecA p-nitrophenyl ß-d-galactoside (pNPGal, Kd = 54 ± 6 µM) demonstrated a 6-fold improved binding of d-Gal proving this approach to be valuable for ligand design in future drug discovery campaigns that aim to generate inhibitors of LecA.

Fluorine NMR Spectroscopy Enables to Quantify the Affinity Between DNA and Proteins in Cell Lysate.

The determination of the binding affinity quantifying the interaction between proteins and nucleic acids is of crucial interest in biological and chemical research. Here, we have made use of site-specific fluorine labeling of the cold shock protein from Bacillus subtilis, BsCspB, enabling to directly monitor the interaction with single stranded DNA molecules in cell lysate. High-resolution 19 F NMR spectroscopy has been applied to exclusively report on resonance signals arising from the protein under study. We have found that this experimental approach advances the reliable determination of the binding affinity between single stranded DNA molecules and its target protein in this complex biological environment by intertwining analyses based on NMR chemical shifts, signal heights, line shapes and simulations. We propose that the developed experimental platform offers a potent approach for the identification of binding affinities characterizing intermolecular interactions in native surroundings covering the nano-to-micromolar range that can be even expanded to in cell applications in future studies.

Evaluation of fluorine-19 magnetic resonance imaging of the lungs using octafluorocyclobutane in a rat model.

PURPOSE: To test octafluorocyclobutane (OFCB) as an inhalation contrast agent for fluorine-19 MRI of the lung, and to compare the image quality of OFCB scans with perfluoropropane (PFP) scans THEORY AND METHODS: After normalizing for the number of signal averages, a theoretical comparison between the OFCB signal-to-noise ratio (SNR) and PFP SNR predicted the average SNR advantage of 90% using OFCB during gradient echo imaging. The OFCB relaxometry was conducted using single-voxel spectroscopy and spin-echo imaging. A comparison of OFCB and PFP SNRs was performed in vitro and in vivo. Five healthy Sprague-Dawley rats were imaged during single breath-hold and continuous breathing using a Philips Achieva 3.0T MRI scanner (Philips, Andover, MA). The scan time was constant for both gases. Statistical comparison between PFP and OFCB scans was conducted using a paired t test and by calculating the Bayes factor. RESULTS: Spin-lattice (T1 ) and effective spin-spin ( T2∗ ) relaxation time constants of the pure OFCB gas were determined as 28.5 ± 1.2 ms and 10.5 ± 1.8 ms, respectively. Mixing with 21% of oxygen decreased T1 by 30% and T2∗ by 20%. The OFCB in vivo images showed 73% higher normalized SNR on average compared with images acquired using PFP. The statistical significance was shown by both paired t test and calculated Bayes factors. The experimental results agree with theoretical calculations within the error of the relaxation parameter measurements. CONCLUSION: The quality of the lung images acquired using OFCB was significantly better compared with PFP scans. The OFCB images had higher a SNR and were artifact-free.

A2A adenosine receptor functional states characterized by 19F-NMR.

The human proteome contains 826 G protein-coupled receptors (GPCR), which control a wide array of key physiological functions, making them important drug targets. GPCR functions are based on allosteric coupling from the extracellular orthosteric drug binding site across the cell membrane to intracellular binding sites for partners such as G proteins and arrestins. This signaling process is related to dynamic equilibria in conformational ensembles that can be observed by NMR in solution. A previous high-resolution NMR study of the A2A adenosine receptor (A2AAR) resulted in a qualitative characterization of a network of such local polymorphisms. Here, we used 19F-NMR experiments with probes at the A2AAR intracellular surface, which provides the high sensitivity needed for a refined description of different receptor activation states by ensembles of simultaneously populated conformers and the rates of exchange among them. We observed two agonist-stabilized substates that are not measurably populated in apo-A2AAR and one inactive substate that is not seen in complexes with agonists, suggesting that A2AAR activation includes both induced fit and conformational selection mechanisms. Comparison of A2AAR and a constitutively active mutant established relations between the 19F-NMR spectra and signaling activity, which enabled direct assessment of the difference in basal activity between the native protein and its variant.

Cascaded Multiresponsive Self-Assembled 19F MRI Nanoprobes with Redox-Triggered Activation and NIR-Induced Amplification.

Molecular probes featuring promising capabilities including specific targeting, high signal-to-noise ratio, and in situ visualization of deep tissues are in great demand for tumor diagnosis and therapy. 19F magnetic resonance imaging (MRI) techniques incorporating stimuli-responsive probes are anticipated to be highly beneficial for specific detection and imaging of tumors because of negligible background and deep tissue penetration. Herein, we report a cascaded multiresponsive self-assembled nanoprobe, which enables sequential redox-triggered and near-infrared (NIR) irradiation-induced 19F MR signal activation/amplification for sensing and imaging. Specifically, we designed and synthesized a cascaded multiresponsive 19F-bearing nanoprobe based on the self-assembly of amphiphilic redox-responsive 19F-containing polymers and NIR-absorbing indocyanine green (ICG) molecules. It could realize the activation of 19F signals in the reducing tumor microenvironment and subsequent signal amplification via the photothermal process. This stepwise two-stage activation/amplification of 19F signals was validated by 19F NMR and MRI both in vitro and in vivo. The multiresponsive 19F nanoprobes capable of cascaded 19F signal activation/amplification and photothermal effect exertion can provide accurate sensing and imaging of tumors.

Fluorine-19 Magnetic Resonance Imaging for Detection of Amyloid ß Oligomers Using a Keto Form of Curcumin Derivative in a Mouse Model of Alzheimer's Disease.

Recent evidence suggests that the formation of soluble amyloid ß (Aß) aggregates with high toxicity, such as oligomers and protofibrils, is a key event that causes Alzheimer's disease (AD). However, understanding the pathophysiological role of such soluble Aß aggregates in the brain in vivo could be difficult due to the lack of a clinically available method to detect, visualize, and quantify soluble Aß aggregates in the brain. We had synthesized a novel fluorinated curcumin derivative with a fixed keto form, named as Shiga-Y51, which exhibited high selectivity to Aß oligomers in vitro. In this study, we investigated the in vivo detection of Aß oligomers by fluorine-19 (19F) magnetic resonance imaging (MRI) using Shiga-Y51 in an APP/PS1 double transgenic mouse model of AD. Significantly high levels of 19F signals were detected in the upper forebrain region of APP/PS1 mice compared with wild-type mice. Moreover, the highest levels of Aß oligomers were detected in the upper forebrain region of APP/PS1 mice in enzyme-linked immunosorbent assay. These findings suggested that 19F-MRI using Shiga-Y51 detected Aß oligomers in the in vivo brain. Therefore, 19F-MRI using Shiga-Y51 with a 7 T MR scanner could be a powerful tool for imaging Aß oligomers in the brain.

Single Fluorinated Agent for Multiplexed 19 F-MRI with Micromolar Detectability Based on Dynamic Exchange.

The weak thermal polarization of nuclear spins limits the sensitivity of MRI, even for MR-sensitive nuclei as fluorine-19. Therefore, despite being the source of inspiration for the development of background-free MRI for various applications, including for multiplexed imaging, the inability to map very low concentrations of targets using 19 F-MRI raises the need to further enhance this platform's capabilities. Here, we employ the principles of CEST-MRI in 19 F-MRI to obtain a 900-fold signal amplification of a biocompatible fluorinated agent, which can be presented in a "multicolor" fashion. Capitalizing on the dynamic interactions in host-guest supramolecular assemblies in an approach termed GEST, we demonstrate that an inhalable fluorinated anesthetic can be used as a single 19 F-probe for the concurrent detection of micromolar levels of two targets, with potential in vivo translatability. Further extending GEST with new designs could expand the applicability of 19 F-MRI to the mapping of targets that have so-far remained non-detectable.

Performance of compressed sensing for fluorine-19 magnetic resonance imaging at low signal-to-noise ratio conditions.

PURPOSE: To examine the performance of compressed sensing (CS) in reconstructing low signal-to-noise ratio (SNR) 19 F MR signals that are close to the detection threshold and originate from small signal sources with no a priori known location. METHODS: Regularization strength was adjusted automatically based on noise level. As performance metrics, root-mean-square deviations, true positive rates (TPRs), and false discovery rates were computed. CS and conventional reconstructions were compared at equal measurement time and evaluated in relation to high-SNR reference data. 19 F MR data were generated from a purpose-built phantom and benchmarked against simulations, as well as from the experimental autoimmune encephalomyelitis mouse model. We quantified the signal intensity bias and introduced an intensity calibration for in vivo data using high-SNR ex vivo data. RESULTS: Low-SNR 19 F MR data could be reliably reconstructed. Detection sensitivity was consistently improved and data fidelity was preserved for undersampling and averaging factors of α = 2 or = 3. Higher α led to signal blurring in the mouse model. The improved TPRs at α = 3 were comparable to a 2.5-fold increase in measurement time. Whereas CS resulted in a downward bias of the 19 F MR signal, Fourier reconstructions resulted in an unexpected upward bias of similar magnitude. The calibration corrected signal-intensity deviations for all reconstructions. CONCLUSION: CS is advantageous whenever image features are close to the detection threshold. It is a powerful tool, even for low-SNR data with sparsely distributed 19 F signals, to improve spatial and temporal resolution in 19 F MR applications.

An iterative sparse deconvolution method for simultaneous multicolor 19 F-MRI of multiple contrast agents.

PURPOSE: 19 F-MRI is gaining widespread interest for cell tracking and quantification of immune and inflammatory cells in vivo. Different fluorinated compounds can be discriminated based on their characteristic MR spectra, allowing in vivo imaging of multiple 19 F compounds simultaneously, so-called multicolor 19 F-MRI. We introduce a method for multicolor 19 F-MRI using an iterative sparse deconvolution method to separate different 19 F compounds and remove chemical shift artifacts arising from multiple resonances. METHODS: The method employs cycling of the readout gradient direction to alternate the spatial orientation of the off-resonance chemical shift artifacts, which are subsequently removed by iterative sparse deconvolution. Noise robustness and separation was investigated by numerical simulations. Mixtures of fluorinated oils (PFCE and PFOB) were measured on a 7T MR scanner to identify the relation between 19 F signal intensity and compound concentration. The method was validated in a mouse model after intramuscular injection of fluorine probes, as well as after intravascular injection. RESULTS: Numerical simulations show efficient separation of 19 F compounds, even at low signal-to-noise ratio. Reliable chemical shift artifact removal and separation of PFCE and PFOB signals was achieved in phantoms and in vivo. Signal intensities correlated excellently to the relative 19 F compound concentrations (r-2 = 0.966/0.990 for PFOB/PFCE). CONCLUSIONS: The method requires minimal sequence adaptation and is therefore easily implemented on different MRI systems. Simulations, phantom experiments, and in-vivo measurements in mice showed effective separation and removal of chemical shift artifacts below noise level. We foresee applicability for simultaneous in-vivo imaging of 19 F-containing fluorine probes or for detection of 19 F-labeled cell populations.