Guillermier C, Poczatek JC, Taylor WR, Steinhauser ML. Quantitative imaging of deuterated metabolic tracers in biological tissues with nanoscale secondary ion mass spectrometry. International Journal of Mass Spectrometry. 2017.Abstract
In the field of secondary ion mass spectrometry at nanometer scale (NanoSIMS), configuration of parallel detectors to routinely measure isotope ratios in sub-100 nm domains brings classical stable isotope tracer studies from the whole tissue level down to the suborganelle level. Over the past decade, the marriage of stable isotope tracers with NanoSIMS has been applied to a range of fundamental biological questions that were largely inaccessible by other means. Although multiplexed measurement of different stable isotope tracers is feasible, in practice there remains a gap in the current analytical capacity to efficiently measure stable isotopes commonly utilized in tracer studies. One such example is the measurement of deuterated tracers. The most obvious approach to measuring deuterium/hydrogen isotope ratios is at mass 2/1. However, the radius of the magnetic sector limits concomitant measurement of other masses critical to multiplexed exploration of biological samples. Here we determine the experimental parameters to measure deuterated tracers in biological samples using the C2H polyatomic ion species (C2D/C2H) while operating the NanoSIMS at a reduced Mass Resolving Power of 14,000. Through control of the sputtering parameters, we demonstrate that there is an analytical window during which the C2D/C2H isotope ratio can be measured with sufficient precision for biological studies where the degree of D-labeling is typically well above natural abundance. We provide validation of this method by comparing the C2D measurement of D-water labeling in the murine small intestine relative to measurements of native D/H conducted in the same analytical fields. Additional proof-of-concept demonstrations include measurement of D-water, D-glucose, and D-thymidine in biological specimens. Therefore, this study provides a practical template for deuterium-based tracer studies in biological systems.
Guillermier C, Fazeli PK, Kim S, Lun M, Zuflacht JP, Milian J, Steinhauser ML. Imaging mass spectrometry demonstrates age-related decline in human adipose plasticity. JCI Insight. 2017;2 (5).Abstract
Quantification of stable isotope tracers has revealed the dynamic state of living tissues. A new form of imaging mass spectrometry quantifies isotope ratios in domains much smaller than a cubic micron, enabling measurement of cell turnover and metabolism with stable isotope tracers at the single-cell level with a methodology we refer to as multi-isotope imaging mass spectrometry. In a first-in-human study, we utilize stable isotope tracers of DNA synthesis and de novo lipogenesis to prospectively measure cell birth and adipocyte lipid turnover. In a study of healthy adults, we elucidate an age-dependent decline in new adipocyte generation and adipocyte lipid turnover. A linear regression model suggests that the aging effect could be mediated by a decline in insulin-like growth factor-1 (IGF-1). This study therefore establishes a method for measurement of cell turnover and metabolism in humans with subcellular resolution while implicating the growth hormone/IGF-1 axis in adipose tissue aging.
Kumar S, Rajagopalan S, Sarkar P, Dorward DW, Peterson ME, Liao H-S, Guillermier C, Steinhauser ML, Vogel SS, Long EO. Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses. Molecular Cell [Internet]. 2016;62 (1) :21-33. Publisher's VersionAbstract

The inhibitory function of killer cell immunoglobulin-like receptors (KIR) that bind HLA-C and block activation of human natural killer (NK) cells is dependent on zinc. We report that zinc induced the assembly of soluble KIR into filamentous polymers, as detected by electron microscopy, which depolymerized after zinc chelation. Similar KIR filaments were isolated from lysates of cells treated with zinc, and membrane protrusions enriched in zinc were detected on whole cells by scanning electron microscopy and imaging mass spectrometry. Two independent mutations in the extracellular domain of KIR, away from the HLA-C binding site, impaired zinc-driven polymerization and inhibitory function. KIR filaments formed
spontaneously, without the addition of zinc, at functional inhibitory immunological synapses of NK cells with HLA-C + cells. Adding to the recent paradigm of signal transduction through higher order molecular assemblies, zinc-induced polymerization of inhibitory KIR represents an unusual mode of signaling
by a receptor at the cell surface.

Bailey AP, Koster G, Guillermier C, Hirst EMA, MacRae JI, Lechene CP, Postle AD, Gould AP. Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila. Cell [Internet]. 2015;163 (2) :340–353. Publisher's VersionAbstract
Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts.
Steinhauser ML, Guillermier C, Wang M, Lechene CP. Approaches to increasing analytical throughput of human samples with multi-isotope imaging mass spectrometry. Surface and Interface Analysis [Internet]. 2014;46 (S1) :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.
Thiery-Lavenant G, Guillermier C, Wang M, Lechene C. Detection of immunolabels with multi-isotope imaging mass spectrometry. Surface and Interface Analysis [Internet]. 2014;46 (S1) :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 MD, Moy J, Wang M, Guillermier C, Poczatek JC, Turck CW, Lechene C. Effect of an antidepressant on mouse hippocampus protein turnover analyzed by MIMS. Surface and Interface Analysis [Internet]. 2014;46 (S1) :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 ML, Guillermier C, Wang M, Lechene CP. Quantifying cell division with deuterated water and multi-isotope imaging mass spectrometry (MIMS). Surface and Interface Analysis [Internet]. 2014;46 (S1) :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, Guillermier C, Loss O, Poczatek JC, Lechene C. Quantitative imaging of inositol distribution in yeast using multi-isotope imaging mass spectrometry (MIMS). Surface and Interface Analysis [Internet]. 2014;46 (S1) :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 SS, Guillermier C, Wang M, Poczatek JC, Suzuki N, Loscalzo J, Lechene C. Quantitative imaging of selenoprotein with multi-isotope imaging mass spectrometry (MIMS). Surface and Interface Analysis [Internet]. 2014;46 (S1) :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 ML, Lechene CP. Quasi-simultaneous acquisition of nine secondary ions with seven detectors on NanoSIMS50L: application to biological samples. Surface and Interface Analysis [Internet]. 2014;46 (S1) :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.
Enikolopov G, Guillermier C, Wang M, Trakimas L, Steinhauser ML, Lechene C. Brain stem cell division and maintenance studied using multi-isotope imaging mass spectrometry (MIMS). Surface and Interface Analysis [Internet]. 2014;46 :140–143. Publisher's Version
Kim SM, Lun M, Wang M, Senyo SE, Guillermier C, Patwari P, Steinhauser ML. Loss of White Adipose Hyperplastic Potential Is Associated with Enhanced Susceptibility to Insulin Resistance. Cell Metabolism [Internet]. 2014;20 :1049–1058. Publisher's VersionAbstract

Fat mass expansion occurs by adipocyte hypertrophy or recruitment of differentiating adipocyte progenitors, the relative balance of which may impact systemic metabolism. We measured adipogenesis in murine subcutaneous (sWAT) and visceral white adipose tissue (vWAT) using stable isotope methodology and then modeled adipocyte turnover. Birth and death rates were similar within depots; however, turnover was higher in vWAT relative to sWAT. In juvenile mice, obesity increased adipogenesis, but in adults, this was only seen in vWAT after prolonged high-fat feeding. Statistical modeling suggests differentiation of adipocyte progenitors without an accompanying self-renewing division step may partially explain the age-dependent decline in hyperplastic potential. Additional metabolic interrogation of obese mice demonstrated an association between adipocyte turnover and insulin sensitivity. These data therefore identify adipocyte hypertrophy as the dominant mechanism of adult fat mass expansion and support the paradoxical concept that metabolic disease ensues due to a failure of adipose tissue plasticity.

Brismar H, Aperia A, Westin L, Moy J, Wang M, Guillermier C, Poczatek JC, Lechene C. Study of protein and RNA in dendritic spines using multi-isotope imaging mass spectrometry. Surface and Interface Analysis [Internet]. 2014;46 :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.

Senyo SE, Steinhauser ML, Pizzimenti CL, Yang VK, Cai L, Wang M, Wu T-D, Guerquin-Kern J-L, Lechene CP, Lee RT. Mammalian heart renewal by pre-existing cardiomyocytes. Nature [Internet]. 2013;493 (7432) :433–6. Publisher's VersionAbstract
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 ML, Lechene CP. Quantitative imaging of subcellular metabolism with stable isotopes and multi-isotope imaging mass spectrometry. Seminars in cell & developmental biology [Internet]. 2013 :1–7. Publisher's VersionAbstract
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.
Steinhauser ML, Bailey AP, Senyo SE, Guillermier C, Perlstein TS, Gould AP, Lee RT, Lechene CP. Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism. Nature [Internet]. 2012;481 (7382) :516–9. Publisher's VersionAbstract
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.
Zhang D-S, Piazza V, Perrin BJ, Rzadzinska AK, Poczatek JC, Wang M, Prosser HM, Ervasti JM, Corey DP, Lechene CP. Multi-isotope imaging mass spectrometry reveals slow protein turnover in hair-cell stereocilia. Nature [Internet]. 2012;481 (7382) :520–4. Publisher's VersionAbstract
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.
Gormanns P, Reckow S, Poczatek CJ, Turck CW, Lechene C. Segmentation of multi-isotope imaging mass spectrometry data for semi-automatic detection of regions of interest. PloS one [Internet]. 2012;7 (2) :e30576. Publisher's VersionAbstract
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\_ImageJ.php.
Lechene CP, Lee GY, Poczatek CJ, Toner M, Biggers JD. 3D multi-isotope imaging mass spectrometry reveals penetration of 18O-trehalose in mouse sperm nucleus. PloS one [Internet]. 2012;7 (8) :e42267. Publisher's VersionAbstract
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.