National Resource for Imaging Mass Spectrometry

Cambridge, MA, United States

National Resource for Imaging Mass Spectrometry

Cambridge, MA, United States
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Steinhauser M.L.,Harvard University | Guillermier C.,Harvard University | Wang M.,National Resource for Imaging Mass Spectrometry | Lechene C.P.,Harvard University
Surface and Interface Analysis | Year: 2014

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. Copyright © 2014 John Wiley & Sons, Ltd.


Thiery-Lavenant G.,Harvard University | Guillermier C.,Harvard University | Wang M.,National Resource for Imaging Mass Spectrometry | Lechene C.,Harvard University
Surface and Interface Analysis | Year: 2014

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 1.4nm 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. Copyright © 2014 John Wiley & Sons, Ltd.


Steinhauser M.L.,Harvard University | Guillermier C.,Harvard University | Wang M.,National Resource for Imaging Mass Spectrometry | Lechene C.P.,Harvard University
Surface and Interface Analysis | Year: 2014

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. Copyright © 2014 John Wiley & Sons, Ltd.


Brismar H.,Astrid Lindgren Childrens Hospital | Aperia A.,Astrid Lindgren Childrens Hospital | Westin L.,Astrid Lindgren Childrens Hospital | Moy J.,National Resource for Imaging Mass Spectrometry | And 4 more authors.
Surface and Interface Analysis | Year: 2014

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. Copyright © 2014 John Wiley & Sons, Ltd.


PubMed | Harvard University, National Resource for Imaging Mass Spectrometry and Astrid Lindgren Childrens Hospital
Type: Journal Article | Journal: Surface and interface analysis : SIA | Year: 2015

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


PubMed | National Resource for Imaging Mass Spectrometry. and Harvard University
Type: Journal Article | Journal: Surface and interface analysis : SIA | Year: 2015

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


PubMed | National Resource for Imaging Mass Spectrometry. and Harvard University
Type: Journal Article | Journal: Surface and interface analysis : SIA | Year: 2015

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

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