Publications

2014
Steinhauser, M.L., et al., 2014. {Approaches to increasing analytical throughput of human samples with multi-isotope imaging mass spectrometry}. Surface and Interface Analysis, 46, pp.165–168. Publisher's VersionAbstract
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. 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 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 and imaged, and their coordinates were 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.
Enikolopov, G., et al., 2014. {Brain stem cell division and maintenance studied using multi-isotope imaging mass spectrometry (MIMS)}. Surface and Interface Analysis, 46, pp.140–143. Publisher's VersionAbstract
New neurons are continuously produced from neural stem cells in specific regions of the adult brain of animals and humans. In the hippocampus, a region crucial for cognitive function, neurogenesis responds to a multitude of extrinsic stimuli; emerging evidence indicates that it may be important for behavior, pathophysiology, brain repair, and response to drugs. We have developed an approach to identify and quantify the cellular targets of pro- and anti-neurogenic stimuli, based on reporter transgenic mouse lines in which neural stem and progenitor cells or their progeny are marked by fluorescent proteins. Here, we demonstrate the feasibility of using multi-isotope imaging mass spectrometry for studying adult neurogenesis.
Thiery-Lavenant, G., et al., 2014. {Detection of immunolabels with multi-isotope imaging mass spectrometry}. Surface and Interface Analysis, 46, pp.147–149. Publisher's VersionAbstract
We have developed a method that combines the use of stable isotopes, multi-isotope imaging mass spectrometry (MIMS) and antibody. We began with using well-established antibodies, anti-actin and anti-synaptophysin, in mouse intestinal cells. We extended the method to an immunogold assay to specifically localize Ribeye, a major protein component of retina synaptic ribbons, or to localize a synaptic vesicle-containing protein, synaptophysin. Both are localized in presynaptic nerve terminal of photoreceptors cells in retina. Our results show that by MIMS analysis of the Au signal, we can directly identify antibodies tagged with non amplified 1.4 nm gold nanoparticles. They also demonstrate that the gold nanoparticle-tagged antibodies do not dilute the 15 N/14 N signal used for measuring protein turnover. Thus, we can simultaneously and directly use MIMS to measure protein turnover and to identify cell type or specific protein.
Filiou, M.D., et al., 2014. {Effect of an antidepressant on mouse hippocampus protein turnover analyzed by MIMS}. Surface and Interface Analysis, 46, pp.144–146. Publisher's VersionAbstract
Although antidepressants have been used in the treatment of affective disorders for over 50 years, the precise mechanism of their action remains unknown. Treatment regimens are based by and large on empirical parameters and characterized by a trial and error scheme. A better understanding of the mechanisms involved in antidepressant drug response is of fundamental importance for the development of new compounds that have a higher success rate and specificity. In order to elucidate the molecular pathways involved in the action of antidepressants, we wish to identify brain areas, cell types and organelles that are targeted by antidepressant treatment in mice. Multi-isotope imaging mass spectrometry allows a quantitative approach to this analysis, enabling us to delineate antidepressant effect on protein synthesis in the brain at single cell and organelle resolution. In these experiments, we obtained a global analysis of protein turnover in the hippocampus dentate gyrus and in the Cornu Ammonis regions, together with a subcellular analysis in the granular cells and others.
Steinhauser, M.L., et al., 2014. {Quantifying cell division with deuterated water and multi-isotope imaging mass spectrometry (MIMS)}. Surface and Interface Analysis, 46, pp.161–164. Publisher's VersionAbstract
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.
Saiardi, A., et al., 2014. {Quantitative imaging of inositol distribution in yeast using multi-isotope imaging mass spectrometry (MIMS)}. Surface and Interface Analysis, 46, pp.169–172. Publisher's VersionAbstract
Despite the widely recognized importance of the several species of inositol polyphosphates in cell biology, inositol has not been successfully imaged and quantified inside cells using traditional spectrophotometry. Multi-isotope imaging mass spectrometry (MIMS) technology, however, has facilitated direct imaging and measurement of cellular inositol. After pulsing cells with inositol labeled with the stable isotope Carbon-13 (13C), the label was detected in subcellular volumes by MIMS. The tridimensional localization of 13C within the cell illustrated cellular distribution and local accumulation of inositol. In parallel, we performed control experiments with 13C-glucose to compare a different 13C distribution pattern. Because many functions recently attributed to inositol polyphosphates are localized in the nucleus, we analyzed its relative nuclear concentration. We engineered yeast with human thymidine permease and viral thymidine kinase then fed them with 15N-thymidine. This permitted direct analysis of the nuclear DNA through the detection of the 15N isotopic signal. We found practically no co-localization between inositol signal (13C-isotope) and nuclear signal (15N-isotope). The 13C-tag (inositol) accumulation was highest at the plasma membrane and in cytoplasmic domains. In time-course labeling experiments performed with wild-type (WT) yeast or modified yeast unable to synthesize inositol from glucose (ino1$Δ$), the halftime of labeled inositol accumulation was \~{}1 h in WT and longer in ino1$Δ$. These studies should serve as a template to study metabolism and physiological role of inositol using genetically modified yeasts.
Tang, S.S., et al., 2014. {Quantitative imaging of selenoprotein with multi-isotope imaging mass spectrometry (MIMS)}. Surface and Interface Analysis, 46, pp.154–157. Publisher's VersionAbstract
Multi-isotope imaging mass spectrometry (MIMS) allows high-resolution quantitative imaging of protein and nucleic acid synthesis at the level of a single cell using stable isotope labels. We employed MIMS to determine the compartmental localization of selenoproteins tagged with stable isotope selenium compounds in human aortic endothelial cells (HAEC), and to compare the efficiency of labeling (to determine the ideal selenium source) from these compounds: [82Se]-selenite, [77Se]-seleno-methionine, and [76Se]-methyl-selenocysteine. We found that all three selenium isotopes appear to be localized in the nucleus as well as in the cytoplasm. For MIMS detection, we compared freeze-drying to thin layer versus thin sectioning for sample preparation. MIMS provides a unique and novel way to dissect selenoprotein synthesis in cells.
Guillermier, C., Steinhauser, M.L. & Lechene, C.P., 2014. {Quasi-simultaneous acquisition of nine secondary ions with seven detectors on NanoSIMS50L: application to biological samples}. Surface and Interface Analysis, 46, pp.150–153. Publisher's VersionAbstract
We employed a method of electrostatic peak switching allowing for the quasi-simultaneous measurement of 16O, 18O, C2H, C2D, 12C14N, 13C14N, 12C15N, P, and S with the NanoSIMS 50L instrument to derive ratios for D/H, 13C/12C, 18O/16O, and 15N/14N from biological samples. This approach involves two steps: (i) derivation of the D/H ratio from measurements of C2D and C2H and (ii) switching of the voltage on deflection plates located in front of two detectors. The method is reliable and easy to set up compared with the magnetic peak-switching mode usually used to perform this type of analysis.
Brismar, H., et al., 2014. {Study of protein and RNA in dendritic spines using multi-isotope imaging mass spectrometry}. Surface and Interface Analysis, 46, pp.158–160. Publisher's VersionAbstract
The classical view of neuronal protein synthesis is that proteins are made in the cell body and then transported to their functional sites in the dendrites and the dendritic spines. Indirect evidence, however, suggests that protein synthesis can directly occur in the distal dendrites, far from the cell body. We are developing protocols for dual labeling of RNA and proteins using 15 N-uridine and 18O- or 13C-leucine pulse chase in cultured neurons to identify and localize both protein synthesis and fate of newly synthesized proteins. Pilot experiments show discrete localization of both RNA and newly synthesized proteins in dendrites, close to dendritic spines. We have for the first time directly imaged and measured the production of proteins at the subcellular level in the neuronal dendrites, close to the functional sites, the dendritic spines. This will open a powerful way to study neural growth and synapse plasticity in health and disease.
2013
Senyo, S.E., et al., 2013. {Mammalian heart renewal by pre-existing cardiomyocytes.}. Nature, 493, pp.433–6. PubMedAbstract
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.
Steinhauser, M.L. & Lechene, C.P., 2013. {Quantitative imaging of subcellular metabolism with stable isotopes and multi-isotope imaging mass spectrometry.}. Seminars in cell & developmental biology, pp.1–7. PubMedAbstract
Multi-isotope imaging mass spectrometry (MIMS) is the quantitative imaging of stable isotope labels in cells with a new type of secondary ion mass spectrometer (NanoSIMS). The power of the methodology is attributable to (i) the immense advantage of using non-toxic stable isotope labels, (ii) high resolution imaging that approaches the resolution of usual transmission electron microscopy and (iii) the precise quantification of label down to 1 part-per-million and spanning several orders of magnitude. Here we review the basic elements of MIMS and describe new applications of MIMS to the quantitative study of metabolic processes including protein and nucleic acid synthesis in model organisms ranging from microbes to humans.
2012
Lechene, C.P., et al., 2012. {3D multi-isotope imaging mass spectrometry reveals penetration of 18O-trehalose in mouse sperm nucleus.}. PloS one, 7, pp.e42267. PubMedAbstract
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.
Steinhauser, M.L., et al., 2012. {Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism.}. Nature, 481, pp.516–9. PubMedAbstract
Mass spectrometry with stable isotope labels has been seminal in discovering the dynamic state of living matter, 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 submicrometre resolution. 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 labelling mice with (15)N-thymidine from gestation until post-natal week 8, we find no (15)N label retention by dividing small intestinal crypt cells after a four-week chase. In adult mice administered (15)N-thymidine pulse-chase, we find that proliferating crypt cells dilute the (15)N 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 haematopoietic system. These studies show that MIMS provides high-resolution quantification of stable isotope labels that cannot be obtained using other techniques and that is broadly applicable to biological and medical research.
Faculty of 1000 Review Rating: Exceptional
Zhang, D.-S., et al., 2012. {Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia.}. Nature, 481, pp.520–4. PubMedAbstract
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 $\beta$-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 $\beta$-actin-GFP we bleached fiducial lines across hair bundles, but they did not move in 6 days. When we stopped expression of $\beta$- or $\gamma$-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.
Faculty of 1000 Review Rating: Exceptional
Gormanns, P., et al., 2012. {Segmentation of multi-isotope imaging mass spectrometry data for semi-automatic detection of regions of interest.}. PloS one, 7, pp.e30576. PubMedAbstract
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/NRIMSİmageJ.php.
2007
Kuypers, M.M.M., 2007. {Microbiology. Sizing up the uncultivated majority.}. Science (New York, N.Y.), 317, pp.1510–1. ScienceAbstract
A new imaging technique allows the metabolic activity of single microbial cells to be quantified in environmental samples.
Lechene, C.P., et al., 2007. {Quantitative imaging of nitrogen fixation by individual bacteria within animal cells.}. Science (New York, N.Y.), 317, pp.1563–6. PubMedAbstract
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.
Faculty of 1000 Review Rating: Exceptional
2006
Weitzman, J.B., 2006. {Imaging with isotopes: high resolution and quantitation.}. Journal of biology, 5, pp.17. PubMed
Lechene, C., et al., 2006. {High-resolution quantitative imaging of mammalian and bacterial cells using stable isotope mass spectrometry.}. Journal of biology, 5(6), pp.20. PubMedAbstract
Secondary-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.
Faculty of 1000 Review Rating: Exceptional
McMahon, G., Glassner, B.J. & Lechene, C.P., 2006. {Quantitative imaging of cells with multi-isotope imaging mass spectrometry (MIMS)—Nanoautography with stable isotope tracers}. Applied Surface Science, 252, pp.6895–6906. ScienceDirectAbstract
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.
simsxv-050927final2.pdf