Multi-isotope Imaging Mass Spectrometry Research Articles

Approaches to increasing analytical throughput of human samples with multi-isotope imaging mass spectrometry.

Multi-isotope imaging mass spectrometry (MIMS) combines stable isotope tracers with the quantitative imaging of NanoSIMS ion microscopy. With extensive safety precedent, use of stable isotopes in MIMS applications opens the possibility of studying a wide array of biological questions in humans[1]. Here we describe a series of approaches to increase the effective analytical throughput for detecting rare nuclear labeling events with MIMS. At the level of sample preparation, cells in suspension were either smeared at high density or pelleted cells were embedded and sectioned to reach nuclear depth. Presputtering conditions were optimized for each cell type to ensure the reproducible sampling of nuclei. Adipose tissue posed a different challenge as the large volume of adipocytes results in an obligatorily low density of nuclei in any given plane. Before introducing samples to the NanoSIMS instrument, all nuclei were fluorescently stained, imaged, and their coordinates recorded, allowing automated analysis of fields that contained at least one nucleus and therefore minimizing analysis of dead space. These data emphasize unique challenges posed by human studies, where both ethical and practical issues may limit the administration of stable isotope labels for prolonged periods of time as may be necessary to achieve high labeling frequencies in cells that divide infrequently.

Quantifying cell division with deuterated water and multi-isotope imaging mass spectrometry (MIMS).

Cell division is commonly quantified by the administration of nucleotide labels that are incorporated by the nucleotide salvage pathway. A new approach uses precursors of the de novo nucleotide synthesis pathway, such as labeled water or glucose. Because such precursors are not specific for DNA synthesis, studies utilizing this approach have analyzed isolated genomic DNA to exclude nonspecific background labeling. We hypothesized that pulse-chase administration of stable isotope labeled water would result in sufficient nuclear labeling to enable discrimination of recently divided cells by quantitative ion microscopy. We administered deuterated (D)-water and 15N-thymidine to mice concurrently, guided by the rationale that 15N-thymidine incorporation would serve as a "gold standard" to identify dividing cells. We show both qualitatively and quantitatively that dividing cells in the small intestine (15N-labeled) demonstrate a discernable D-signal in the nucleus not observed in undivided cells (15N-unlabled). Correlation with 31P- and 12C15N-:12C14N- images demonstrate preferential localization of 2H labeling in regions of the nucleus with high DNA content as expected of labeling being incorporated during DNA synthesis and cell division. These data support the concept that stable isotope tagged precursors of the de novo nucleotide synthesis pathway can be used in concert with NanoSIMS to study cell division in vivo. A major implication of this study then is the possibility of using stable isotope tagged water and MIMS to study human cell turnover.

Mammalian heart renewal by pre-existing cardiomyocytes

Although recent studies have revealed that heart cells are generated in adult mammals, the frequency of generation and the source of new heart cells are not yet known. Some studies suggest a high rate of stem cell activity with differentiation of progenitors to cardiomyocytes. Other studies suggest that new cardiomyocytes are born at a very low rate, and that they may be derived from the division of pre-existing cardiomyocytes. Here we show, by combining two different pulse-chase approaches--genetic fate-mapping with stable isotope labelling, and multi-isotope imaging mass spectrometry--that the genesis of cardiomyocytes occurs at a low rate by the division of pre-existing cardiomyocytes during normal ageing, a process that increases adjacent to areas of myocardial injury. We found that cell cycle activity during normal ageing and after injury led to polyploidy and multinucleation, but also to new diploid, mononucleate cardiomyocytes. These data reveal pre-existing cardiomyocytes as the dominant source of cardiomyocyte replacement in normal mammalian myocardial homeostasis as well as after myocardial injury.

Abstract 19780: Mammalian Cardiac Renewal by Preexisting Cardiomyocytes

Although recent studies have revealed that adult heart cells are generated after birth in mammals, the frequency and source of these new heart cells is unclear. Here we combined two different pulse-chase approaches--genetic fate-mapping and stable isotope labeling with Multi-isotope Imaging Mass Spectrometry (MIMS)--to determine the origin of cardiomyocytes in adult mice. MIMS allows quantitative imaging of stable isotope reporters in domains smaller than a micron cubed. The nuclear incorporation of a 15N stable isotope-enriched tracer here as 15N-thymidine is detectable with high precision by an increase in the 15N/14N ratio above natural abundance (0.37%) in a pattern resembling chromatin evident in cells having divided during a labeling period. We administered 15N-thymidine for 8 weeks to three age groups of C57BL6 mice starting at day-4 (neonate), at 10-weeks (young adult) and at 22-months (old adult) and extrapolated DNA synthesis yields a yearly rate of 5.5% in the young adult that declines further to 2.6% in advanced aged mice. Fig1a. To evaluate the origin of newly generated cardiomyocytes, we performed 15N-thymidine labeling of double-transgenic MerCreMer/ZEG mice, which we previously developed for genetic lineage mapping. Fig1b. After treatment with 4OH-tamoxifen, we show that DNA synthesis occurs at a low rate in predominantly pre-existing cardiomyocytes [77% expressed GFP, a frequency essentially identical to that of surrounding 15N- cardiomyocytes (15N, 77% vs 15N-, 84%; Fisher’s exact=n.s.)]. Fig1c. Cell cycle activity in cardiomyocytes led to polyploidy and multinucleation, and to new diploid, mononucleated cardiomyocytes [17% of total; predominantly GFP+ (83% vs 82%, p=n.s.)]. Fig1d. Our results support a low rate of cell division of cardiomyocytes as the origin of mammalian adult cardiogenesis and are inconsistent with a high rate of stem cell activity in the normal mammalian myocardium.

3D multi-isotope imaging mass spectrometry reveals penetration of 18O-trehalose in mouse sperm nucleus.

The prevalence of genetically engineered mice in medical research has led to ever increasing storage costs. Trehalose has a significant beneficial effect in preserving the developmental potential of mouse sperm following partial desiccation and storage at temperatures above freezing. Using multi-isotope imaging mass spectrometry, we are able to image and measure trehalose in individual spermatozoa. We provide the first evidence that trehalose penetrates the nucleus of a mammalian cell, permitting tolerance to desiccation. These results have broad implications for long-term storage of mammalian cells.

High-Resolution Multi-isotope Imaging Mass Spectrometry Enables Visualisation of Stem Cell Division and Metabolism

MIMS visualises metabolism: A recent publication by Steinhauser and co-workers presents a novel application of multi-isotope mass spectrometry (MIMS) to visualise physiological metabolism in live mammalian organisms, and validate the "immortal strand hypothesis" of asymmetric chromosomal division of stem cells in the small intestine.

Segmentation of Multi-Isotope Imaging Mass Spectrometry Data for Semi-Automatic Detection of Regions of Interest

Multi-isotope imaging mass spectrometry (MIMS) associates secondary ion mass spectrometry (SIMS) with detection of several atomic masses, the use of stable isotopes as labels, and affiliated quantitative image-analysis software. By associating image and measure, MIMS allows one to obtain quantitative information about biological processes in sub-cellular domains. MIMS can be applied to a wide range of biomedical problems, in particular metabolism and cell fate [1], [2], [3]. In order to obtain morphologically pertinent data from MIMS images, we have to define regions of interest (ROIs). ROIs are drawn by hand, a tedious and time-consuming process. We have developed and successfully applied a support vector machine (SVM) for segmentation of MIMS images that allows fast, semi-automatic boundary detection of regions of interests. Using the SVM, high-quality ROIs (as compared to an expert's manual delineation) were obtained for 2 types of images derived from unrelated data sets. This automation simplifies, accelerates and improves the post-processing analysis of MIMS images. This approach has been integrated into “Open MIMS,” an ImageJ-plugin for comprehensive analysis of MIMS images that is available online at http://www.nrims.hms.harvard.edu/NRIMS_ImageJ.php.

A deeper peek into living organisms

Multi-isotope imaging mass spectrometry (MIMS) is a new and generally applicable method for the study of DNA replication, lipid and protein turnover and cell fate in animals and humans. In a proof-of-principle study, MIMS was used to test the 'immortal strand hypothesis', which proposes that stem cells maintain a master genetic template that is protected from cancer-causing mutations. The hypothesis remains hotly debated, in part because of the difficulties involved in testing it experimentally. Stable isotope incorporation was viewed and measured by MIMS in mammalian intestinal cell division, Drosophila melanogaster lipid metabolism and human lymphopoiesis. In the auditory system, the mechanosensory hair cells of the inner ear convert sound-induced vibrations into electrical signals. The apical surface of a hair cell consists of stereocilia with a core of actin filaments that function as mechanosensors. It has been suggested that these actin filaments are replaced within two to three days by a treadmilling process. Using the newly developed multi-isotope imaging mass spectrometry (MIMS) technique, Zhang et al. quantify protein turnover in hair-cell stereocilia in vivo and find that turnover is slow throughout the stereocilia, except for the tip region, and does not involve a treadmilling process.

Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism

Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter1,2 but is limited to bulk tissues or cells. We developed multi-isotope imaging mass spectrometry (MIMS) that allowed us to view and measure stable isotope incorporation with sub-micron resolution3,4. Here we apply MIMS to diverse organisms, including Drosophila, mice, and humans. We test the “immortal strand hypothesis,” which predicts that during asymmetric stem cell division chromosomes containing older template DNA are segregated to the daughter destined to remain a stem cell, thus insuring lifetime genetic stability. After labeling mice with 15N-thymidine from gestation through post-natal week 8, we find no 15N label retention by dividing small intestinal crypt cells after 4wk chase. In adult mice administered 15N-thymidine pulse-chase, we find that proliferating crypt cells dilute label consistent with random strand segregation. We demonstrate the broad utility of MIMS with proof-of-principle studies of lipid turnover in Drosophila and translation to the human hematopoietic system. These studies show that MIMS provides high-resolution quantitation of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.

Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia

Hair cells of the inner ear are not normally replaced during an animal's life, and must continually renew components of their various organelles. Among these are the stereocilia, each with a core of several hundred actin filaments that arise from their apical surfaces and that bear the mechanotransduction apparatus at their tips. Actin turnover in stereocilia has previously been studied by transfecting neonatal rat hair cells in culture with a β-actin-GFP fusion, and evidence was found that actin is replaced, from the top down, in 2-3 days. Overexpression of the actin-binding protein espin causes elongation of stereocilia within 12-24 hours, also suggesting rapid regulation of stereocilia lengths. Similarly, the mechanosensory 'tip links' are replaced in 5-10 hours after cleavage in chicken and mammalian hair cells. In contrast, turnover in chick stereocilia in vivo is much slower. It might be that only certain components of stereocilia turn over quickly, that rapid turnover occurs only in neonatal animals, only in culture, or only in response to a challenge like breakage or actin overexpression. Here we quantify protein turnover by feeding animals with a (15)N-labelled precursor amino acid and using multi-isotope imaging mass spectrometry to measure appearance of new protein. Surprisingly, in adult frogs and mice and in neonatal mice, in vivo and in vitro, the stereocilia were remarkably stable, incorporating newly synthesized protein at <10% per day. Only stereocilia tips had rapid turnover and no treadmilling was observed. Other methods confirmed this: in hair cells expressing β-actin-GFP we bleached fiducial lines across hair bundles, but they did not move in 6 days. When we stopped expression of β- or γ-actin with tamoxifen-inducible recombination, neither actin isoform left the stereocilia, except at the tips. Thus, rapid turnover in stereocilia occurs only at the tips and not by a treadmilling process.

Seeing and Measuring Stable Isotope Tags in Subcellular Domains with Ion Microscopy

Multi-isotope Imaging Mass Spectrometry (MIMS) is the combination of an ion microscope-secondary ion mass spectrometer with tracer methods and intensive quantitative image analysis. MIMS allows one to image the distribution and to measure the accumulation of molecules labelled with stable or radioactive isotopes within subcellular domains in volume smaller than 1 cubic micron. The lateral resolution is better than 30 nm, and the depth resolution is a few atomic layers. Up to seven quantitative atomic mass images (or tags) can be recorded simultaneously. A whole cell can be studied layer-after-layer and rendered in quantitative 3D.MIMS can provide unique information in almost all fields of biomedical research, particularly in two broad areas: 1. metabolic turnover (protein, lipids and sugars in sub-cellular domains using as precursors for example 15N-labeled amino-acids or 13C labeled fatty acids) 2. cell lineage ( tracking individual cells within large populations by labelingDNA, for example with 15N thymidine for the salvage pathway and 18O for the de novo pathway).Because stable isotopes are integral part of living organisms, they are not toxic. This allows one to label DNA with a stable isotope precursors for period of time that could reach years. And, this allows applications of MIMS to human.

Molecular diversity of bacterioplankton: link to a predictive biogeochemistry of pelagic ecosystems

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 53:39-58 (2008) - DOI: https://doi.org/10.3354/ame01227 AME Special: 'Progress and Perspectives in Aquatic Microbial Ecology: Highlights of the SAME 10, Faro, Portugal, September 2007' Molecular diversity of bacterioplankton: link to a predictive biogeochemistry of pelagic ecosystems Manfred G. Höfle1,*, David L. Kirchman2, Richard Christen3, Ingrid Brettar1 1Department Vaccinology, Helmholtz Centre of Infection Research (HZI), Inhoffenstr. 7, 38124 Braunschweig, Germany 2College of Marine and Earth Studies, University of Delaware, Lewes, Delaware 19958, USA 3Virtual Biology Lab, Centre for Biochemistry, University of Nice Sophia Antipolis & CNRS, Campus Valrose, 06108 Nice, France *Email: mho@gbf.de ABSTRACT: In this paper, we focus on the current understanding of the microbial diversity of bacterioplankton and its link to the biogeochemistry of the pelagic zone of aquatic ecosystems. During the last 2 decades, a large array of molecular approaches has been developed and applied to assess the diversity of bacterioplankton. One key result of the classical molecular approaches, such as community fingerprints and environmental clone libraries, is that the bacterioplankton community contains only a few abundant species and a large number of less abundant to rare members. This is in contrast to soil bacterial communities, where no abundant species are detected and the total number of species is substantially larger. New approaches, such as metagenomics and pyrosequencing, are discussed in terms of their ability to determine the diversity and abundance of the rare members of the community and the biogeochemical potential of the community. Several new approaches, such as stable isotope probing (SIP) and multi-isotope imaging mass spectrometry (MIMS), have been developed to study structure–function relationships of microbial communities. The combination of biogeochemical stimulation experiments with these functional and/or molecular approaches has the potential to elucidate the role of specific microorganisms concerning specific biogeochemical functions. These experimental analyses, together with in situ measurements of molecular and biogeochemical markers, enable the identification of key catalysts of pelagic biogeochemical processes and determination of their regulatory factors. Based on our current understanding of the structure–function links in bacterioplankton, we hypothesize that any local bacterial community comprises 2 parts: the abundant members form a core community that carries out the ongoing biogeochemical functions, and the rare members form a seedbank that provides the genetic potential to respond to any change in the system. Together, both parts form a pan community that is able to mediate the biogeochemical processes in the pelagic ecosystems of the earth. KEY WORDS: Community structure · Pyrosequencing · Metagenomics · Bioinformatics · Comparative genomics · Structure–function links · Drivers · Community theory Full text in pdf format PreviousNextCite this article as: Höfle MG, Kirchman DL, Christen R, Brettar I (2008) Molecular diversity of bacterioplankton: link to a predictive biogeochemistry of pelagic ecosystems. Aquat Microb Ecol 53:39-58. https://doi.org/10.3354/ame01227 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 53, No. 1. Online publication date: September 18, 2008 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2008 Inter-Research.

Quantitative Imaging of Nitrogen Fixation by Individual Bacteria Within Animal Cells

Biological nitrogen fixation, the conversion of atmospheric nitrogen to ammonia for biosynthesis, is exclusively performed by a few bacteria and archaea. Despite the essential importance of biological nitrogen fixation, it has been impossible to quantify the incorporation of nitrogen by individual bacteria or to map the fate of fixed nitrogen in host cells. In this study, with multi-isotope imaging mass spectrometry we directly imaged and measured nitrogen fixation by individual bacteria within eukaryotic host cells and demonstrated that fixed nitrogen is used for host metabolism. This approach introduces a powerful way to study microbes and global nutrient cycles.

Is Membrane Transport of FFA Mediated by Lipid, Protein, or Both?

![Figure][1] J. Patrick Kampf Torrey Pines Institute for Molecular Studies, San Diego, California ![Figure][1] Alan M. Kleinfeld Torrey Pines Institute for Molecular Studies, San Diego, California akleinfeld{at}tpims.org The mechanism of free fatty acid (FFA)

Quantitative imaging of cells with multi-isotope imaging mass spectrometry (MIMS)—Nanoautography with stable isotope tracers

We describe some technical aspects of the application of multi-isotope imaging mass spectrometry (MIMS) to biological research, particularly the use of isotopic tags to localize and measure their incorporation into intracellular compartments. We touch on sample preparation, on image formation, on drift correction and on extraction of quantitative data from isotope ratio imaging. We insist on the wide variety of sample types that can be used, ranging from whole cells prepared directly on Si supports, to thin sections of cells and tissues on Si supports, to ultrathin TEM sections on carbon-coated grid. We attempt to dispel the myth of difficulties in sample preparation, which we view as a needless deterrent to the application of MIMS to the general biological community. We present protocols for the extraction of isotope ratio data from mass images. We illustrate the benefits of using sequential image plane acquisition followed by the application of an autocorrelation algorithm (nanotracking) to remove the effects of specimen drift. We insist on the advantages to display the isotope ratios as hue saturation intensity images.

High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry

BackgroundSecondary-ion mass spectrometry (SIMS) is an important tool for investigating isotopic composition in the chemical and materials sciences, but its use in biology has been limited by technical considerations. Multi-isotope imaging mass spectrometry (MIMS), which combines a new generation of SIMS instrument with sophisticated ion optics, labeling with stable isotopes, and quantitative image-analysis software, was developed to study biological materials.ResultsThe new instrument allows the production of mass images of high lateral resolution (down to 33 nm), as well as the counting or imaging of several isotopes simultaneously. As MIMS can distinguish between ions of very similar mass, such as 12C15N- and 13C14N-, it enables the precise and reproducible measurement of isotope ratios, and thus of the levels of enrichment in specific isotopic labels, within volumes of less than a cubic micrometer. The sensitivity of MIMS is at least 1,000 times that of 14C autoradiography. The depth resolution can be smaller than 1 nm because only a few atomic layers are needed to create an atomic mass image. We illustrate the use of MIMS to image unlabeled mammalian cultured cells and tissue sections; to analyze fatty-acid transport in adipocyte lipid droplets using 13C-oleic acid; to examine nitrogen fixation in bacteria using 15N gaseous nitrogen; to measure levels of protein renewal in the cochlea and in post-ischemic kidney cells using 15N-leucine; to study DNA and RNA co-distribution and uridine incorporation in the nucleolus using 15N-uridine and 81Br of bromodeoxyuridine or 14C-thymidine; to reveal domains in cultured endothelial cells using the native isotopes 12C, 16O, 14N and 31P; and to track a few 15N-labeled donor spleen cells in the lymph nodes of the host mouse.ConclusionMIMS makes it possible for the first time to both image and quantify molecules labeled with stable or radioactive isotopes within subcellular compartments.

In-situ imaging mass spectrometry analysis of melanin granules in the human hair shaft.

The elemental composition of melanin granules and other components of the hair shaft was determined by multi-isotope imaging mass spectrometry, a method with unique advantages for the visualization and quantification of stable isotopes and the elemental composition in study of the fine structure of biologic samples. We mapped and quantified the chemical composition of hair cross-sections using secondary ions generated from naturally occurring 16O, 12C14N, 32S, and 34S with a maximum lateral resolution of 35 nm. Based on these elemental maps of unprecedented resolution we obtained simultaneously the chemical fingerprints and the structural features, such as cuticle, melanin granules, the macro fibrils of the cortex, and small sulfur-rich domains in the medulla, in the hair cross-section. We found an intriguing distribution of 16O, 12C14N, and 32S in melanin granules that we interpret as a highly anisotropic pattern of oxidation.