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Spatial micro-analysis of natural 13C/12C abundance in environmental samples using laser-ablation isotope-ratio-mass spectrometry Andrei Rodionov, Eva Lehndorff, Ciprian C. Stremtan, Willi A Brand, Heinz-Peter Königshoven, and Wulf Amelung Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b00892 • Publication Date (Web): 01 Apr 2019 Downloaded from http://pubs.acs.org on April 3, 2019
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Analytical Chemistry
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Spatial micro-analysis of natural 13C/12C abundance in environmental
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samples using laser-ablation isotope-ratio-mass spectrometry
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Andrei Rodionov1,2*, Eva Lehndorff1,2#, Ciprian C. Stremtan3, Willi A. Brand4, Heinz-
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Peter Königshoven5, Wulf Amelung1
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1 Institute
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Ecology, University of Bonn, Nussallee 13, 53115 Bonn, Germany
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2 Present
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Bayreuth, Germany
of Crop Science and Resource Conservation (INRES), Soil Science and Soil
address: Soil Ecology, University of Bayreuth, Dr.-Hans-Frisch-Str. 1-3, 95448
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3 Teledyne
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4 Max-Planck-Institute
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Jena, Germany
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5 Feinmechanische
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Bonn, Wegeler Str. 12, 53115 Bonn, Germany
CETAC Technologies, 14306 Industrial Rd. Omaha, Nebraska 68144 USA for Biogeochemistry, Beutenberg Campus, P.O. Box 100164, 07701
Werkstatt, Institute of Physical and Theoretical Chemistry, University of
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* Corresponding author (
[email protected]) #shared first authorship
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Abstract
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The stable 13C/12C isotope composition usually varies among different organic materials due to
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isotope fractionation during biochemical synthesis and degradation processes. Here we
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introduce a novel laser-ablation stable isotope-mass-spectrometry methodology (LA-IRMS)
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that allows highly resolved spatial analysis of carbon isotope signatures in solid samples down
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to a spatial resolution of 10 µm. The presented instrumental setup includes in-house-designed
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exchangeable ablation cells (3.8 and 0.4 mL, respectively) and an improved sample gas transfer,
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which allow accurate δ13C measurements of an acryl plate standard down to 0.6 and 0.4 ng of
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ablated carbon, respectively (standard deviation 0.25‰). Initial testing on plant and soil
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samples confirmed that microheterogeneity of their natural
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mapped at a spatial resolution down to 10 µm. The respective δ13C values in soils with C3/C4
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crop sequence history varied by up to 14‰ across a distance of less than 100 µm in soil
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aggregates, while being partly sorted along rhizosphere gradients of < 300 µm from Miscanthus
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plant roots into the surrounding soil. These very first demonstrations point to the appearance of
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very small metabolic hotspots originating from different natural isotope discrimination
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processes, now traceable via LA-IRMS.
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13C/12C
abundance can now be
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Analytical Chemistry
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Introduction
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The stable 13C/12C isotope composition of environmental samples has become a unique tracer
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of carbon assimilation pathways. In plants, its analysis provided key information about the
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assimilation of fossil carbon sources (Suess effect) 1, about differentiated C3 and C4
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photosynthetic pathways 2,3, and gave hints regarding water stress adaptation pathways 4–6. In
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marine and lacustrine environments, the analysis helped to track terrestrial C input and was
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useful for paleoclimate reconstruction 7–9. In terrestrial environments, δ13C analyses were
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indispensable for assessing the mean residence time of soil carbon after C3/C4 vegetation
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changes10,11, for elucidating microbial and faunal food-webs12, and for paleo-environmental
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reconstructions13–16. However, spatially homogeneous C isotope signals were assumed for all
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these studies because it has not been possible to map microscale variation of the 13C/12C isotope
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composition in routine lab analyses down to the natural abundance level. The present
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contribution provides a viable solution to this problem.
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Different instrumental setups using laser ablation (LA) combined with other detectors have
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already been applied on a micrometer scale for measuring bacterial cells and plant and animal
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tissue, as well as to other substances in geological and biological studies17–23. For ablation of
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organic matter, cold Nd:YAG laser ablation systems are recommended24,25. Lasers operating at
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213 nm ensure cold ablation as more traditional 266 nm systems24,25 and thus avoid isotope
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fractionation effects during the ablation process. All previous laser ablation studies relied on
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obtaining spatially resolved signatures from organic solids with relatively large and
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homogeneous organic C contents compared to soil (wood, single hair samples, or pollen grains
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of C3 and C4 plants), yielding ablation spot sizes between 150 and 50 µm22,24,25 and a sensitivity
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between 65 and 24 ng C25,26. Higher spatial resolution and sensitivity are crucial for many
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environmental studies, which have aims such as identifying microbial hotspots in soils and
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sediments, or patchy sequestration of carbon.
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In this contribution we introduce a LA-IRMS system using frequency quintupled 213 nm Q-
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switched Nd:YAG laser with 4 mJ pulse-1;
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Teledyne CETAC Technologies, Omaha, USA). Laser ablation of particulate matter was done
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in newly designed low-volume cells, the ablated particles were collected and transported using
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helium as the carrier gas. Particles were oxidized to CO2 in an oven, then sample gas was
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transferred to a system of two cryo-traps for gas clean-up and peak focus (modified PreCon
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system, see27). Sample gas was then led through a GC-column before it entered the IRMS
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DeltaVplus via a ConFloIV interface (both from ThermoFisher Scientific) for analysis of 13C.
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The challenges were to increase the spatial resolution of the 13C signal by an order of
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magnitude by decreasing the area of ablated spots down to 10 µm, thus responding to a growing
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interest to isotopical characterization of microbial hot spots, organic exudates of roots and
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residues of plants and microbes. Doing so would enable us to reach into areas where the
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utilization of organic materials is physically hindered by, e.g., encrustation processes and
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occlusion within aggregates, and thus limited bioaccessibility.
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Experimental approach
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Ablation system and isotope measurement
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For laser ablation, highly focused laser light pulses are used to remove (ablate) material at a
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spatial resolution of a few micrometers in diameter from discrete areas of virtually any solid
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sample (e.g.,28 and references therein). For the present study, we used a Teledyne LSX-213G2+
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solid state Nd:YAG laser system (CETAC, Omaha, NE, USA) that produces deep UV laser
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radiation at a wavelength of 213 nm. The laser pulse frequency can be variably adjusted;
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frequencies from 10 to 20 Hz proved to be optimal for this application. Furthermore, the laser
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allowed adjustment of the energy output, the ablated spot size and the number of laser shots.
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The standard cell of the LSX213 G2+ laser (two volume active cell HelEx II was replaced with
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an in-house-designed single-volume cell (see Figure 1 − the cell was constructed with an active 4 ACS Paragon Plus Environment
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Analytical Chemistry
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volume of 3.8 cm3 and a total internal volume of 17.2 cm3; for details see Figure S-1 in
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supplementary material). The cell was mounted on an adapted plane mount aligned to the laser’s
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original movable stages, which allowed the sample to be monitored by computer-controlled
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video microscope and internal LED illumination (coaxial, ring, and transmitted light sources).
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As we expected the samples to have very low amounts of C, we also produced a second cell
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with the same design, but with an even smaller active volume of 0.4 cm3 and, therefore, with
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reduced background signal. Both cells are easy to handle and are interchangeable.
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The upper and lower windows of the single-volume cell were made of quartz glass and sealed
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using O-rings (Ø30x2mm; form error: 3/0.5 wedge:
