# Publications

2013
Senyo, S.E., et al., 2013. {Mammalian heart renewal by pre-existing cardiomyocytes.}. Nature, 493, pp.433–6. PubMed Abstract
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. PubMed Abstract
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
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.
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
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
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. Science Abstract
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. PubMed Abstract
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
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. ScienceDirect Abstract
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.
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., et al., 2006. {CN- secondary ions form by recombination as demonstrated using multi-isotope mass spectrometry of 13C- and 15N-labeled polyglycine.}. Journal of the American Society for Mass Spectrometry, 17, pp.1181–7. PubMed Abstract
We have studied the mechanism of formation CN- secondary ions under Cs+ primary ion bombardment. We have synthesized 13C and 15N labeled polyglycine samples with the distance between the two labels and the local atomic environment of the 13C label systematically varied. We have measured four masses in parallel: 12C, 13C, and two of 12C14N, 13C14N, 12C15N, and 13C15N. We have calculated the 13C/12C isotope ratio, and the different combinations of the CN isotope ratios (27CN/26CN, 28CN/27CN, and 28CN/26CN). We have measured a high 13C15N- secondary ion current from the 13C and 15N labeled polyglycines, even when the 13C and 15N labels are separated. By comparing the magnitude of the varied combinations of isotope ratios among the samples with different labeling positions, we conclude the following: CN- formation is in large fraction due to recombination of C and N; the CO double bond decreases the extent of CN- formation compared to the case where carbon is singly bonded to two hydrogen atoms; and double-labeling with 13C and 15N allows us to detect with high sensitivity the molecular ion 13C15N-.
2004
Hallégot, P., Peteranderl, R. & Lechene, C., 2004. {In-situ imaging mass spectrometry analysis of melanin granules in the human hair shaft.}. The Journal of investigative dermatology, 122, pp.381–6. PubMed Abstract
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.
Peteranderl, R. & Lechene, C., 2004. {Measure of carbon and nitrogen stable isotope ratios in cultured cells.}. Journal of the American Society for Mass Spectrometry, 15, pp.478–85. PubMed Abstract
We report the measurement of the natural isotope ratios of nitrogen and carbon in subcellular volumes of individual cells among a population of cultured cells using a multi-isotope imaging mass spectrometer (MIMS), [MIMS is the prototype of the NanoSIMS 50, Cameca, France.] We also measured the nitrogen and carbon isotope ratio in cells after they had been cultured in media enriched with the amino acid glycine labeled with either 13C or 15N. The results demonstrate that 13C/12C and 15N/14N isotope ratios can be measured directly on a subcellular scale. This opens the way for the use of stable isotopes, in particular 15N, as labels to measure the intracellular turnover of biomolecules. Such a capability should help resolve a wide range of biomedical problems.
Kleinfeld, A.M., Kampf, P.J. & Lechene, C., 2004. {Transport of 13C-oleate in adipocytes measured using multi imaging mass spectrometry.}. Journal of the American Society for Mass Spectrometry, 15, pp.1572–80. PubMed Abstract
The mechanism of long chain free fatty acid (FFA) transport across cell membranes is under active investigation. Here we describe the use of multi imaging mass spectrometry (MIMS) to monitor intracellular concentrations of FFA and provide new insight into FFA transport in cultured adipocytes. Cells were incubated with 13C-oleate:BSA and either dried directly or dried after washing with a medium deprived of 13C-oleate:BSA. Cells were analyzed with MIMS using a scanning primary Cs+ ion beam and 12C-, 13C-, 12C14N-, 13C14N-) (or 12C 15N-) were imaged simultaneously. From these quantitative images the values of the 13C/ 12C ratios were determined in the intracellular lipid droplets, in the cytoplasm and outside the 3T3F442A adipocytes. The results indicate that after incubation with 13C-oleate:BSA the droplet 13C/ 12C ratio was 15 +/- 6%. This value is about 14-fold higher than the 13C/ 12C terrestrial ratio (1.12%). After washing the 13C-oleate:BSA, the droplet 13C/ 12C ratios decreased to 1.6 +/- 0.1%, about 40% greater than the natural abundance. Results for washed cells indicate that relatively little FFA was esterified. The unwashed cell results, together with the value of the lipid water partition coefficient, reveal that intracellular unbound FFA (FFAu) concentrations were on average about 4.5-fold greater than the extracellular FFAu concentrations. These results are consistent with the possibility that FFA may be pumped into adipocytes against their electro-chemical potential. This work demonstrates that MIMS can be used to image and quantitate stable isotope labeled fatty acid in intracellular lipid droplets.
2003
Guillermier, C., et al., 2003. {Vacuum bench for the characterization of thermoionization ion sources}. Review of Scientific Instruments, 74, pp.3312. Article Abstract
We have designed a vacuum bench to study the parameters of thermoionization sources with the ultimate goal of obtaining high spatial resolution for biomedical applications of secondary ion mass spectrometry. In the bench, the source ionizer can be directly heated with an electron gun positioned perpendicular to the axis of the ion beam and focused with an optical system including slit lenses and a magnetic sector. The source cross over diameter is measured by forming the image of the source using an Einzel lens at a 1×magnification. The ion beam current is measured in a Faraday cup placed after a movable diaphragm. The temperature of the diverse elements of the ionizer assembly is measured through a mirror with a micropyrometer. Using the vacuum bench with a cesium carbonate source, we measured a 35 μm minimum cross over size, and we calculated a 400 A/cm2/srmaximum brightness. We obtained an intense cesium ion beam when heating the ionizer with the electron gun. The vacuum bench will be used to compare the effect of the heating mode of the ionizer (i.e., indirect by filament electron emission or direct by electron beam) on the brightness of the cesium source, and to develop a thermoionization iodine negative ion source.