Ion Imaging Research Articles

Accurate measurements of the bond dissociation energies of 14N2, 14N15N and 15N2

Accurate determinations of bond dissociation energies (BDEs) and the corresponding enthalpies of formation derived from them are of fundamental importance, while on the other hand also very challenging in chemistry. In this study, we demonstrate a direct experimental scheme for measuring the BDEs of 14N2, 14N15N and 15N2 to an accuracy of a few wavenumbers. The high-resolution quantum-state-selected velocity-mapped ion images for several rotational levels with accurately known spectroscopic term energies slightly above the dissociation limit N(2D5/2, 3/2) + N(2D5/2, 3/2) were obtained for each of the dinitrogen isotopologue molecules. A linear fit between squares of the radii of the quantum-state-selected images and the spectroscopic term energies gives out an accurate measurement of the threshold D0 for dissociating into the channel N(2D5/2, 3/2) + N(2D5/2, 3/2). The BDEs of 4N2, 14N15N and 15N2 into the lowest channel N(4S) + N(4S) are independently determined to be 78688 ± 3, 78705 ± 4 and 78728 ± 4 cm−1, respectively. These independently determined BDEs are found to give a consistent value of De after considering the zero-point energies for each of the isotopologues, implying the reliability of the present measurement. Comparisons with several previous measurements and theoretical calculations are discussed. The experimental scheme demonstrated in this study should be generally applicable for precisely determining the BDEs of many other diatomic molecules.

DNA nanomachine activation and Zn2+ imaging in living cells with single NIR irradiation

Though activatable DNA nanomachines in response to external and internal stimuli successfully improve the temporal and spatial controllability of biosensing and bioimaging, current strategies usually apply different irradiation lights for DNA nanomachine activation and imaging processes. The involvement of short wavelength excitation results in shallow tissue penetration and high background interference. Here we design a photo-locked DNA nanomachine (P-DNA nanomachine) that uses single NIR irradiation for spatiotemporally activating nanomachine operation and metal ion imaging in cancer cells. Upconversion nanoparticles (UCNPs) are modified with energy collector dye FITC, photo-locked DNAzyme and its substrate strands labelled with BHQ1. Part of the UCNPs emission at 450 nm was collected by FITC and quenched by BHQ1 originally. 980 nm irradiation photolyzes PC linker, activates DNAzyme catalytic reaction in the presence of Zn2+, and recovers FITC luminance at 540 nm. The intracellular Zn2+ amount was imaged by quantitatively measuring FITC recovery intensity versus internal standard of UCNPs luminance at 450 nm. The presented NIR activatable P-DNA nanomachine demonstrates effective protection against “false positive” signal from extracellular interference, thus has potential application for in vivo imaging.

Atmospheric-Pressure Infrared Laser-Ablation Plasma-Postionization Mass Spectrometry Imaging of Formalin-Fixed Paraffin-Embedded (FFPE) and Fresh-Frozen Tissue Sections with No Sample Preparation.

Mass spectrometry imaging (MSI) encompasses a powerful suit of techniques which provide spatially resolved atomic and molecular information from almost any sample type. MSI is now widely used in preclinical research to provide insight into metabolic phenotypes of disease. Typically, fresh-frozen tissue preparations are considered optimal for biological MSI and other traditional preservation methods such as formalin fixation, alone or with paraffin embedding (FFPE), are considered less optimal or even incompatible. Due to the prevalence of FFPE tissue storage, particularly for rare and therefore high-value tissue samples, there is substantial motivation for optimizing MSI methods for analysis of FFPE tissue. Here, we present a novel modality, atmospheric-pressure infrared laser-ablation plasma postionization (AP-IR-LA-PPI), with the first proof-of-concept examples of MSI for FFPE and fresh-frozen tissues, with no post-sectioning sample preparation. We present ion images from FFPE and fresh tissues in positive and negative ion modes. Molecular annotations (via the Metaspace annotation engine) and on-tissue MS/MS provide additional confidence that the detected ions arise from a broad range of metabolite and lipid classes from both FFPE and fresh-frozen tissues.

Crossed Molecular Beam Study of the H + HD → H2 + D Reaction at 0.60 and 1.26 eV Using the Near-Threshold Ionization Velocity Map Ion Imaging

By using the 1 + 1' near-threshold ionization velocity map ion imaging technique, state-to-state reactive differential cross sections have been measured for the H + HD → H2 + D reaction. High-resolution images of the D products, with the rotational states of the H2 co-products clearly resolved, were acquired at the collision energies of 0.60 and 1.26 eV, respectively. It is found that the angular distribution is predominantly backward-scattering at the collision energy of 0.60 eV. However, at 1.26 eV, where the collision energy is higher, the angular distribution becomes forward-backward-scattering. Notably, at both collision energies, the main peaks of backward-scattered products gradually shift from backward toward sideways direction as the rotational quantum number of H2 increases. Moreover, in the forward direction, fast angular oscillations, which are induced by specific partial waves have also been observed at 1.26 eV. These features show a strong correlation between the product states and angular distributions and also indicate the unique role of partial waves in quantum reactive scattering.

A near-infrared photoacoustic probe for specific detection of fluoride ion in vivo

Fluoride is an essential trace element in the human body and plays an essential role in various physiological processes. However, fluoride ion detection in vivo remains challenging due to the lack of probes that can enter the organism and emit at the near infrared region (NIR). Herein, we developed a boron-dipyrromethene (BODIPY)-based NIR probe that takes advantage of the high resolution and deep penetration of photoacoustic imaging, which can produce high NIR absorption at 803 nm in the presence of fluoride ions, resulting in a strong photoacoustic signal in the NIR region. In addition, the probe exhibits high selectivity for fluoride ions. In view of these outstanding features, the probe was then successfully applied to fluoride ion imaging in living mice, proving that a probe is a promising tool for fluoride ion detection in vitro and in vivo.

High-Throughput Nano-DESI Mass Spectrometry Imaging of Biological Tissues Using an Integrated Microfluidic Probe.

Nanospray desorption electrospray mass spectrometry imaging (nano-DESI MSI) enables quantitative mapping of hundreds of molecules in biological samples with minimal sample pretreatment. We have recently developed an integrated microfluidic probe (iMFP) for nano-DESI MSI. Herein, we describe an improved design of the iMFP for the high-throughput imaging of tissue sections. We increased the dimensions of the primary and spray channels and optimized the spray voltage and solvent flow rate to obtain a stable operation of the iMFP at both low and high scan rates. We observe that the sensitivity, molecular coverage, and spatial resolution obtained using the iMFP do not change to a significant extent as the scan rate increases. Using a scan rate of 0.4 mm/s, we obtained high-quality images of mouse uterine tissue sections (scan area: 3.2 mm × 2.3 mm) in only 9.5 min and of mouse brain tissue (scan area: 7.0 mm × 5.4 mm) in 21.7 min, which corresponds to a 10-15-fold improvement in the experimental throughput. We have also developed a quantitative metric for evaluating the quality of ion images obtained at different scan rates. Using this metric, we demonstrate that the quality of nano-DESI MSI data does not degrade substantially with an increase in the scan rate. The ability to image biological tissues with high throughput using iMFP-based nano-DESI MSI will substantially speed up tissue mapping efforts.

Recombination between 13C and 2H to Form Acetylide (13C22H-) Probes Nanoscale Interactions in Lipid Bilayers via Dynamic Secondary Ion Mass Spectrometry: Cholesterol and GM1 Clustering.

Although it is thought that there is lateral heterogeneity of lipid and protein components within biological membranes, probing this heterogeneity has proven challenging. The difficulty in such experiments is due to both the small length scale over which such heterogeneity can occur, and the significant perturbation resulting from fluorescent or spin labeling on the delicate interactions within bilayers. Atomic recombination during dynamic nanoscale secondary ion imaging mass spectrometry (NanoSIMS) is a non-perturbative method for examining nanoscale bilayer interactions. Atomic recombination is a variation on conventional NanoSIMS imaging, whereby an isotope on one molecule combines with a different isotope on another molecule during the ionization process, forming an isotopically enriched polyatomic ion in a distance-dependent manner. We show that the recombinant ion, 13C22H-, is formed in high yield from 13C- and 2H-labeled lipids. The low natural abundance of triply labeled acetylide also makes it an ideal ion to probe GM1 clusters in model membranes and the effects of cholesterol on lipid-lipid interactions. We find evidence supporting the cholesterol condensation effect as well as the presence of nanoscale GM1 clusters in model membranes.

The TAM-associated STIM1I484R mutation increases ORAI1 channel function due to a reduced STIM1 inactivation break and an absence of microtubule trapping

Tubular aggregate myopathy (TAM) is a progressive skeletal muscle disease associated with gain-of-function mutations in the ER Ca2+ sensor STIM1 that mediates store-operated Ca2+ entry (SOCE) across the Ca2+-release-activated (CRAC) Ca2+ channel ORAI1. A frameshift mutation in STIM1 inactivation domain, STIM1I484R, was identified in a TAM patient and reported to decrease SOCE. Using ion imaging and electrophysiology, we show that the STIM1I484R mutation instead renders STIM1 constitutively active. In ion imaging experiments, STIM1I484R was less efficient than native STIM1 when expressed alone but enhanced SOCE and increased basal Ca2+ and Mn2+ influx when expressed together with ORAI1. In patch-clamp recordings, STIM1I484R generated larger pre-activated CRAC currents lacking slow Ca2+-dependent inhibition (SCDI). STIM1I484R was pre-recruited in plasma membrane clusters when co-expressed with ORAI1, as were mutants truncated at the frameshift residue or lacking EB-1-binding, which recapitulated STIM1I484R gain-of-function. When expressed alone in human primary myoblasts, STIM1I484R was pre-recruited in large clusters and increased basal Ca2+ entry. These observations establish that STIM1I484R confers a gain of CRAC channel function due to the loss of critical inhibitory C-terminal domains that prevent STIM1 binding to ORAI1, enable STIM1 trapping by microtubules, and mediate SCDI, providing a mechanistic explanation for the muscular defects of TAM patients bearing this mutation.

Sequential paired covariance for improved visualization of mass spectrometry imaging datasets.

Untargeted analyses in mass spectrometry imaging produce hundreds of ion images representing spatial distributions of biomolecules in biological tissues. Due to the large diversity of ions detected in untargeted analyses, normalization standards are often difficult to implement to account for pixel‐to‐pixel variability in imaging studies. Many normalization strategies exist to account for this variability, but they largely do not improve image quality. In this study, we present a new approach for improving image quality and visualization of tissue features by application of sequential paired covariance (SPC). This approach was demonstrated using previously published tissue datasets such as rat brain and human prostate with different biomolecules like metabolites and N‐linked glycans. Data transformation by SPC improved ion images resulting in increased smoothing of biological features compared with commonly used normalization approaches.

Quantitative determination of sn-positional phospholipid isomers in MSn using silver cationization

Glycerophospholipids are one of the fundamental building blocks for life. The acyl chain connectivity to the glycerol backbone constitutes different sn-positional isomers, which have great diversity and importance for biological function. However, to fully realize their impact on function, analytical techniques that can identify and quantify sn-positional isomers in chemically complex biological samples are needed. Here, we utilize silver ion cationization in combination with tandem mass spectrometry (MSn) to identify sn-positional isomers of phosphatidylcholine (PC) species. In particular, a labile carbocation is generated through a neutral loss (NL) of AgH, the dissociation of which provides diagnostic product ions that correspond to acyl chains at the sn-1 or sn-2 position. The method is comparable to currently available methods, has a sensitivity in the nM–µM range, and is compatible with quantitative imaging using mass spectrometry in MS4. The results reveal a large difference in isomer concentrations and the ion images show that the sn-positional isomers PC 18:1_18:0 are homogeneously distributed, whereas PC 18:1_16:0 and PC 20:1_16:0 show distinct localizations to sub-hippocampal structures.Graphical abstract

Desorption Electrospray Ionization Mass Spectrometry Imaging Illustrates the Quality Characters of Isatidis Radix

For a long history, herbal medicines have made significant contributions to human health all around the world. However, the exploration of an effective approach to illustrate their inner quality remains a challenge. So, it is imperative to develop new methods and technologies to characterize and identify quality markers of herbal medicines. Taking Isatidis Radix, the dried root of Isatis indigotica as an example, desorption electrospray ionization (DESI), in combination with quadrupole-time-of-flight mass spectrometry (Q-TOF/MS), was applied in this work for the first time to reveal the comprehensive spatial distribution of metabolites and, further, to illustrate quality characters of this herbal medicine. After simple pretreatment, 102 metabolites including alkaloids, sulfur-containing compounds, phenylpropanoids, nucleosides, amino acids, organic acids, flavonoids, phenols, terpenes, saccharides, peptides, and sphingolipids were characterized, some of which were successfully localized and visualized in the transverse section of the root. Based on the ion images, samples with different quality characters were distinguished unambiguously by the pattern recognition method of orthogonal partial least squares discrimination analysis (OPLS-DA). Simultaneously, 11 major influencing components exerting higher ion intensities in superior samples were identified as the potential quality markers of Isatidis Radix. Desorption electrospray ionization (DESI) mass spectrometry imaging (MSI), together with chemometric analysis could not only improve the understanding of the plant biology of herbal medicines but also be beneficial in the identification of quality markers, so as to carry out better quality control of herbal medicines.

Abstract 979: Analysis of metabolic and genetic changes in human colorectal cancer and colorectal metastatic tissue by combined NanoString technology and mass spectrometry imaging (MSI)

Abstract Introduction: The development of normal colon tissue to carcinoma can progress through several pathways, which exhibit different clinical, pathological and genetic features. Increasing evidence indicates that tumor development and metastases are not solely driven by genetic alterations but also the tumor microenvironment which is associated with changes in metabolic processes. While many studies show the importance of gene expression profiling in cancer progression, little is known about how this correlates with altered lipid metabolism at metastatic sites within the peritoneal cavity. NanoString nCounter provides specific and sensitive genetic profiling data identifying up to 800 genes simultaneously from small amounts of RNA. For biomolecule analysis, MSI is a powerful tool as it enables the rapid identification of phenotype-dependent MS signatures from tissues with little sample preparation. The aim of this study was to investigate the characteristics of secondary tumors at different metastatic sites compared to the primary tumors using a combination of immunohistochemistry, NanoString analysis and Desorption Electrospray Ionization (DESI) MSI. Methods: 12 fresh-frozen human colorectal adenocarcinoma and metastases samples were cut into 12μm sections. DESI-MSI (Xevo G2-XS, Waters) was performed in both polarities with 50μm spatial resolution. SCiLS Lab (Bruker) software were used for data analysis. Histologically-stained (H&E) adjacent sections were used for microscopy. For genomic analysis, RNA was extracted using AllPrep DNA/RNA micro kit from 20μm sections. Following this, RNA abundance was measured using Qubit fluorimeter. Lastly, mRNA hybridisation, detection and scanning was performed on a NanoString nCounter mRNA Gene Expression system and analysed using nSolver 4.0 software. Results: H&E staining allowed the visualisation of different tissue histopathologies. These differences are echoed in selected DESI-MSI ion images. DESI spectra from distinct regions of the tissue show differences in biomolecule expression especially for phospholipids (m/z 600-900). Ions such as m/z 812 correlated with the non-cancerous region whereas others such as m/z 861 were only observed in the cancerous regions of the tissue. Gene expression analysis showed concordance between primary and metastatic tumors such as in RAS/RAF genes. Others, such as IL-6 expression was seen to upregulated in the metastatic sites compared to the primary tumor. Multivariate analysis enabled identification of biomolecules representative of different tissue phenotypes, with the data showing putative MS biomarkers associated with genetic mutations. Conclusion: Results confirmed the advantage of the combined techniques to reveal spatial correlations between changes in lipid metabolism, tumor heterogeneity and gene expression. Citation Format: Yasmin Shanneik, Emrys Jones, Michal Smiga, Bipasha Chakrabarty, Omer Aziz, Steven Pringle, Kaye Williams, Adam McMahon. Analysis of metabolic and genetic changes in human colorectal cancer and colorectal metastatic tissue by combined NanoString technology and mass spectrometry imaging (MSI) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 979.

Recent development in chemosensor probes for the detection and imaging of zinc ions: a systematic review

Recent development in chemosensor probes for the detection and imaging of zinc ions: a systematic review

Spatial Mapping of Plant N-Glycosylation Cellular Heterogeneity Inside Soybean Root Nodules Provided Insights Into Legume-Rhizobia Symbiosis.

Although ubiquitously present, information on the function of complex N-glycan posttranslational modification in plants is very limited and is often neglected. In this work, we adopted an enzyme-assisted matrix-assisted laser desorption/ionization mass spectrometry imaging strategy to visualize the distribution and identity of N-glycans in soybean root nodules at a cellular resolution. We additionally performed proteomics analysis to probe the potential correlation to proteome changes during symbiotic rhizobia-legume interactions. Our ion images reveal that intense N-glycosylation occurs in the sclerenchyma layer, and inside the infected cells within the infection zone, while morphological structures such as the cortex, uninfected cells, and cells that form the attachment with the root are fewer N-glycosylated. Notably, we observed different N-glycan profiles between soybean root nodules infected with wild-type rhizobia and those infected with mutant rhizobia incapable of efficiently fixing atmospheric nitrogen. The majority of complex N-glycan structures, particularly those with characteristic Lewis-a epitopes, are more abundant in the mutant nodules. Our proteomic results revealed that these glycans likely originated from proteins that maintain the redox balance crucial for proper nitrogen fixation, but also from enzymes involved in N-glycan and phenylpropanoid biosynthesis. These findings indicate the possible involvement of Lewis-a glycans in these critical pathways during legume-rhizobia symbiosis.

Observation of rotationally dependent fine-structure branching ratios near the predissociation threshold N(2D5/2,3/2) + N(2D5/2,3/2) of 14N2.

Photofragment spin-orbit fine-structure branching ratios have long been predicted to depend on the rotational quantum number J' by theory near the dissociation thresholds of several diatomic molecules, while this has rarely been observed in any photodissociation experiments yet. Here, we measured the fine-structure branching ratios N(2D5/2)/N(2D3/2) produced in the N(2D5/2,3/2) + N(2D5/2,3/2) channel at the b'1Σu +(v = 20) state of 14N2 by using our vacuum ultraviolet (VUV)-pump-VUV-probe time-sliced velocity-mapped ion imaging setup. It is found that 14N2 almost exclusively dissociates into the spin-orbit channel N(2D5/2) + N(2D3/2) at low rotational levels and gradually approaches the statistical or diabatic limit by distributing all possible spin-orbit channels at higher rotational levels. The strongly rotationally dependent fine-structure branching ratios should be due to the increasing strength of nonadiabatic Coriolis interaction among various dissociative states in the so-called "recoupling zone" as J' increases. They are supposed to provide unprecedented information on the near threshold photodissociation dynamics of 14N2.

Fragmentation Dynamics of Fluorene Explored Using Ultrafast XUV-Vis Pump-Probe Spectroscopy

We report on the use of extreme ultraviolet (XUV, 30.3 nm) radiation from the Free-electron LASer in Hamburg (FLASH) and visible (Vis, 405 nm) photons from an optical laser to investigate the relaxation and fragmentation dynamics of fluorene ions. The ultrashort laser pulses allow to resolve the molecular processes occurring on the femtosecond timescales. Fluorene is a prototypical small polycyclic aromatic hydrocarbon (PAH). Through their infrared emission signature, PAHs have been shown to be ubiquitous in the universe, and they are assumed to play an important role in the chemistry of the interstellar medium. Our experiments track the ionization and dissociative ionization products of fluorene through time-of-flight mass spectrometry and velocity-map imaging. Multiple processes involved in the formation of each of the fragment ions are disentangled through analysis of the ion images. The relaxation lifetimes of the excited fluorene monocation and dication obtained through the fragment formation channels are reported to be in the range of a few tens of femtoseconds to a few picoseconds.

Carbon dots for multicolor cell imaging and ultra-sensitive detection of multiple ions in living cells: One Stone for multiple Birds

Given the significant impact of ions on environment pollution and human health, it is urgently needed to establish effective and convenient ion detection approaches, particularly in living cells. In this paper, we constructed multicolor N-doped-carbon dots (mPD-CDs) by facile one-step hydrothermal carbonization of m-phenylenediamine (mPD). mPD-CDs were successfully deployed for multicolor cellular imaging for animal cells, fungi, and bacteria in a wash-free way with high photostability and satisfactory biocompability. Moreover, mPD-CDs can be used as a fluorescent sensing probe for ultrasensitive detection of both iodide ion (I−) and typical heavy metals such as cadmium (Cd2+), copper (Cu2+), mercury (Hg2+), gadolinium (Gd3+), ferrous ion (Fe2+), Zinc (Zn2+), and ferric ion (Fe3+). This is the first report using CDs as optical sensing probe for the detection of Gd3+, and for detection of Fe3+ with fluorescence “turn on”. More significantly, with these versatile and fascinating properties, we applied mPD-CDs for intracellular ion detection in living cells like Hep G2 and S. cerevisiae, and zebra fish. Altogether, mPD-CDs displayed great potential for multicolor cell imaging and the multiple ion detection in vitro and in vivo, presenting a promising strategy for in-situ ultrasensitive sensing of multiple metal ions in the environment and the biological systems.

Solar Flux Influence on the In‐Situ Plasma Density at Topside Ionosphere Measured by Swarm Satellites

AbstractIn this study, we perform the first comprehensive comparison of ion density (Ni) in the topside ionosphere measured by the Langmuir probe (LP) and faceplate (FP) of the thermal ion imager on board Swarm satellites. Our results show a systematic difference between the LP and FP derived Ni values, and the systematic difference shows prominent dependences on solar flux, local time, and season. Although both Ni datasets show generally good linear regression with electron density (Ne) measurements from the incoherent scatter radar (ISR) located at Jicamarca, the Ni derived from LP shows an additional dependence on the solar flux, while such a dependence cannot be seen in the FP‐derived Ni. We suggest that the solar flux dependence of LP‐derived Ni is related to the ion compositions change at Swarm altitude, which has not been properly accounted for in the LP processing algorithm. More light ions (e.g., H+), diffusing down from the plasmasphere to the Swarm altitude, seem to cause the overestimation of Ni from LP during low solar activity. A linear relation between the Swarm LP‐derived Ni and ISR Ne is derived, and such a function is recommended to be implemented into further updates of the Swarm LP plasma density data.

Fluorescent Indicators For Biological Imaging of Monatomic Ions.

Monatomic ions play critical biological roles including maintaining the cellular osmotic pressure, transmitting signals, and catalyzing redox reactions as cofactors in enzymes. The ability to visualize monatomic ion concentration, and dynamic changes in the concentration, is essential to understanding their many biological functions. A growing number of genetically encodable and synthetic indicators enable the visualization and detection of monatomic ions in biological systems. With this review, we aim to provide a survey of the current landscape of reported indicators. We hope this review will be a useful guide to researchers who are interested in using indicators for biological applications and to tool developers seeking opportunities to create new and improved indicators.

Surface engineered bimetallic gold/silver nanoclusters for in situ imaging of mercury ions in living organisms.

Chemical sensing for the sensitive and reliable detection of mercury(II) ions (Hg2+) is of great importance in environmental protection, food safety, and biomedical applications. Due to the bio-enrichment property of Hg2+ in organisms, it is particularly meaningful to develop an effective tool that can in situ and rapidly monitor the level of Hg2+ in living organisms. In this work, we report ligand functionalized gold-silver bimetallic nanoclusters with bright red fluorescence as intracellular probes for imaging Hg2+ in living cells and zebrafish. The bimetallic nanoclusters of DTT-GSH@Au/AgNCs (DG-Au/AgNCs) with strong fluorescence that benefited from the synergistic effect of Au and Ag atoms were obtained through a one-pot synthesis method, incorporating glutathione (GSH) and dithiothreitol (DTT) as the reducers and functionalized ligands. Attractively, the bright red fluorescence of DG-Au/AgNCs could be rapidly and selectively quenched by Hg2+ within 1min with a very low detection limit of 1.01nM. Additionally, DG-Au/AgNCs had a great advantage in the detection of Hg2+ in living cells and zebrafish owing to its notably strong red fluorescence at 665nm, which could avoid effectively auto-fluorescence interference from the organism. Such easily prepared bimetallic fluorescent nanoclusters would be expected to provide a noninvasive and sensitive approach in the detection of heavy metals in situ for environmental protection.