{"id":15993,"date":"2020-11-01T21:07:13","date_gmt":"2020-11-02T05:07:13","guid":{"rendered":"https:\/\/www.darkenergybiosphere.org\/?post_type=publication&#038;p=15993"},"modified":"2020-11-15T14:16:18","modified_gmt":"2020-11-15T22:16:18","slug":"nanosims-sample-preparation-decreases-isotope-enrichment-magnitude-variability-and-implications-for-single%e2%80%90cell-rates-of-microbial-activity","status":"publish","type":"publication","link":"https:\/\/www.darkenergybiosphere.org\/publication\/nanosims-sample-preparation-decreases-isotope-enrichment-magnitude-variability-and-implications-for-single%e2%80%90cell-rates-of-microbial-activity\/","title":{"rendered":"NanoSIMS sample preparation decreases isotope enrichment: magnitude, variability and implications for single\u2010cell rates of microbial activity"},"content":{"rendered":"<p>The activity of individual microorganisms can be measured within environmental samples by detecting uptake of isotope\u2010labelled substrates using nano\u2010scale secondary ion mass spectrometry (nanoSIMS). Recent studies have demonstrated that sample preparation can decrease\u00a0<sup>13<\/sup>C and\u00a0<sup>15<\/sup>N enrichment in bacterial cells, resulting in underestimates of activity. Here, we explore this effect with a variety of preparation types, microbial lineages and isotope labels to determine its consistency and therefore potential for correction. Specifically, we investigated the impact of different protocols for fixation, nucleic acid staining and catalysed reporter deposition fluorescence\u00a0<i>in situ<\/i>\u00a0hybridization (CARD\u2010FISH) on &gt;14\u2009500 archaeal and bacterial cells (<i>Methanosarcina acetivorans<\/i>,\u00a0<i>Sulfolobus acidocaldarius<\/i>\u00a0and\u00a0<i>Pseudomonas putida<\/i>) enriched in\u00a0<sup>13<\/sup>C,\u00a0<sup>15<\/sup>N,\u00a0<sup>18<\/sup>O,\u00a0<sup>2<\/sup>H and\/or\u00a0<sup>34<\/sup>S. We found these methods decrease isotope enrichments by up to 80% \u2013 much more than previously reported \u2013 and that the effect varies by taxa, growth phase, isotope label and applied protocol. We make recommendations for how to account for this effect experimentally and analytically. We also re\u2010evaluate published nanoSIMS datasets and revise estimated microbial turnover times in the marine subsurface and nitrogen fixation rates in pelagic unicellular cyanobacteria. When sample preparation is accounted for, cell\u2010specific rates increase and are more consistent with modelled and bulk rates.<\/p>\n","protected":false},"template":"","tags":[],"publication_type":[19],"_links":{"self":[{"href":"https:\/\/www.darkenergybiosphere.org\/wp-json\/wp\/v2\/publication\/15993"}],"collection":[{"href":"https:\/\/www.darkenergybiosphere.org\/wp-json\/wp\/v2\/publication"}],"about":[{"href":"https:\/\/www.darkenergybiosphere.org\/wp-json\/wp\/v2\/types\/publication"}],"wp:attachment":[{"href":"https:\/\/www.darkenergybiosphere.org\/wp-json\/wp\/v2\/media?parent=15993"}],"wp:term":[{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.darkenergybiosphere.org\/wp-json\/wp\/v2\/tags?post=15993"},{"taxonomy":"publication_type","embeddable":true,"href":"https:\/\/www.darkenergybiosphere.org\/wp-json\/wp\/v2\/publication_type?post=15993"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}