Article pubs.acs.org/ac
A New Radio Frequency Plasma Oxygen Primary Ion Source on Nano Secondary Ion Mass Spectrometry for Improved Lateral Resolution and Detection of Electropositive Elements at Single Cell Level Julien Malherbe,*,† Florent Penen,† Marie-Pierre Isaure,† Julia Frank,‡ Gerd Hause,§ Dirk Dobritzsch,‡ Etienne Gontier,∥ François Horréard,⊥ François Hillion,⊥ and Dirk Schaumlöffel*,†
Anal. Chem. 2016.88:7130-7136. Downloaded from pubs.acs.org by UNIV OF SOUTH DAKOTA on 09/05/18. For personal use only.
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Université de Pau et des Pays de l’Adour, CNRS, Institut des Sciences Analytiques et de Physico-Chimie pour l’Environnement et les Matériaux (IPREM), UMR 5254, 64000 Pau, France ‡ Martin-Luther Universität Halle-Wittenberg, Institute for Biochemistry and Biotechnology, Plant Biochemistry, Weinbergweg 22, 06120 Halle (Saale), Germany § Martin-Luther-Universität Halle-Wittenberg, Biozentrum, Weinbergweg 22, 06120 Halle (Saale), Germany ∥ Université de Bordeaux, Bordeaux Imaging Center UMS 3420 CNRS - US4 INSERM, Pôle d’Imagerie Électronique, 146 Rue Léo Saignat, 33076 Bordeaux, France ⊥ CAMECA, 29 Quai des Grésillons, 92622 Gennevilliers, France S Supporting Information *
ABSTRACT: An important application field of secondary ion mass spectrometry at the nanometer scale (NanoSIMS) is the detection of chemical elements and, in particular, metals at the subcellular level in biological samples. The detection of many trace metals requires an oxygen primary ion source to allow the generation of positive secondary ions with high yield in the NanoSIMS. The duoplasmatron oxygen source is commonly used in this ion microprobe but cannot achieve the same quality of images as the cesium primary ion source used to produce negative secondary ions (C−, CN−, S−, P−) due to a larger primary ion beam size. In this paper, a new type of an oxygen ion source using a rf plasma is fitted and characterized on a NanoSIMS50L. The performances of this primary ion source in terms of current density and achievable lateral resolution have been characterized and compared to the conventional duoplasmatron and cesium sources. The new rf plasma oxygen source offered a net improvement in terms of primary beam current density compared to the commonly used duoplasmatron source, which resulted in higher ultimate lateral resolutions down to 37 nm and which provided a 5−45 times higher apparent sensitivity for electropositive elements. Other advantages include a better long-term stability and reduced maintenance. This new rf plasma oxygen primary ion source has been applied to the localization of essential macroelements and trace metals at basal levels in two biological models, cells of Chlamydomonas reinhardtii and Arabidopsis thaliana.
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currently equipped with two different primary ion sources: a thermal ionization cesium positive ion source 5 and a duoplasmatron source used to produce negative oxygen ions.6 A Cs+ source preferentially generates negative secondary ions (e.g., for electronegative elements such as C, O, S, Si) whereas the duoplasmatron source favors the formation of positive secondary ions (e.g., for alkali and transition metals). The duoplasmatron principle relies on a low-pressure magnetically confined plasma formed by an electrical discharge between a hollow cathode and an anode kept at different potentials. The
econdary ion mass spectrometry (SIMS) is a multidisciplinary analytical technique which relies on sputtering of ions from a solid surface by a primary ion beam and subsequent analysis of the ejected secondary ions by a mass spectrometer. Owing to the different possible combinations of primary ion sources (e.g., Ga+, Bi+, Cs+, O−, and C60+) and mass spectrometers (quadrupole, time-of-flight, double sector), a wide range of applications is achievable in fields spanning from material science to biology and geochemistry. One specific type of SIMS instrument, called NanoSIMS, has been designed to act as a “scanning ion microprobe” which can investigate the elemental and isotopic composition of materials at the lateral submicrometer scale. Since its introduction in the 1990s,1−3 the high spatial resolution of the NanoSIMS opened numerous new research possibilities.4 The instrument is © 2016 American Chemical Society
Received: March 23, 2016 Accepted: June 11, 2016 Published: June 12, 2016 7130
DOI: 10.1021/acs.analchem.6b01153 Anal. Chem. 2016, 88, 7130−7136
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Analytical Chemistry Cs+ source relies on the thermal ionization of Cs atoms in vapor state in contact with a hot metallic surface followed by ion extraction with an electric field. For the NanoSIMS, the most important requirement of the source is to achieve the highest beam current densities in the smallest primary ion beam size on the sample in order to attain high lateral resolution combined with high sensitivity. One crucial ion source characteristic is its brightness which is expressed in mA × cm−2 × sr−1. The minimum beam size on the sample, i.e., the highest lateral resolution of the analysis, is also dependent on the energy dispersion of the respective ion beams6,7 through the chromatic aberration contribution of the final objective lens. For the Cs+ source this energy dispersion is less than 1 eV6 whereas for the duoplasmatron source it is in the order of 15 eV.6 As a consequence, a poorer lateral resolution for the duoplasmatron source is observed compared to the cesium source, mainly due to the lower brightness of the duoplasmatron source. Moreover, frequent maintenance is a known inconvenience for the duoplasmatron source especially when used for the production of negative ions.8 Therefore, alternative primary ion sources should be developed for improving the generation of positive secondary ions in NanoSIMS, e.g., for the detection of alkali and transition metals with high sensitivity and high lateral resolution. The use of rf sources for SIMS applications have been described since the beginnings of SIMS.9 In rf sources, the plasma is inductively coupled to an rf antenna and different designs have been tested.10 However, to date one of the major drawbacks of rf plasma based sources for microfocusing application was the large energy dispersion of ions (30−500 eV) which caused chromatic aberration and therefore prevented the formation of a sufficiently small primary ion beam suitable for NanoSIMS applications. An important application field of NanoSIMS is the detection of chemical elements at the subcellular level in biological samples.11 Probing the distribution of macro and trace elements at the subcellular level is one of the major challenges in cell biology. Because of the above-described limitations of the duoplasmatron ion source, to date, the majority of NanoSIMS works in this field has been performed using the cesium primary ion source, e.g., for the detection of H, C, N (via CN−) O, P, and S in biological cells. However, for the detection of many trace metals, a cesium ion source is not suitable. Thus, an oxygen primary ion source is necessary allowing the generation of positive secondary ions in NanoSIMS. In this paper, a new design of rf plasma oxygen source was fitted and characterized on a NanoSIMS 50L. The performances of this source in terms of current density and achievable lateral resolution have been compared to the conventional duoplasmatron and cesium sources. This new oxygen ion source has been applied to localize essential macroelements and trace metals in cells of two well accepted model organisms: C. reinhardtii (unicellular green algae) and A. thaliana (the most thoroughly studied flowering plant).
source, virtually no maintenance is required as the sputtering of the source parts is negligible, i.e., ions leaving the plasma strikes surrounding walls with low energies (15−20 eV). The generated plasma ions have an axial energy spread in the 5−6 eV range. Figure 1 describes the main parts of the source. The coil is capacitively coupled by impedance to the rf source thus forming
Figure 1. Simplified sketch of the different parts of the rf plasma oxygen primary ion source with (1) rf source, (2) impedence-matching capacitors, (3) gas inlet, (4) dielectric plasma tube, (5) coil, (6) variable-strength electromagnet, (7) extraction and skimmer block, (8) plasma, and (9) ion beam.
a helical rf antenna whose axis coincides with that of the plasma tube. The application of a rf current to the antenna will induce ionization of the oxygen gas and the generation of an oxygen plasma. The impedance matching circuit enables efficient transfer of power to the plasma. Because of capacitive coupling between the coil and the plasma, the rf currents circulating through the coil can generate undesirable radial electric field which can modulate the plasma and ultimately cause an undesirable spread of the beam energy. The source design enables the formation of regions with virtually no plasma fluctuation by adjusting the phase shift across the antenna. The energy of ions extracted is therefore not modulated by the rf antenna thus minimizing energy spread. The movement of electrons around the plasma makes them collide with atoms to produce ions. The plasma can therefore generate a high ion density with relatively low thermal ion energy. The electromagnet placed between the end of the coil and the extraction aperture and lenses is designed to reduce electron diffusion and loss to the wall of the plasma tube thus increasing plasma density in the extraction region. The beam is extracted from the lower region of the plasma, close to the electromagnet, where the concentration of positive or negative ion species is higher. The source diameter was specified by the manufacturer to be about 35−50 μm and the brightness to be 100 mA × cm−2 × sr−1 for O− ions and a source voltage of 8 kV. For comparison, the source diameter of the duoplasmatron source is 300 μm and the brightness around 10−20 mA × cm−2 × sr−1for O− ions and a source voltage of 8 kV. NanoSIMS. The Hyperion rf plasma source was fitted to a NanoSIMS 50L (Cameca, Gennevilliers, France) as primary oxygen ion source above the conventional Cs+ primary ion
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EXPERIMENTAL SECTION Apparatus. Overview of the Oxygen rf Plasma Ion Source. The Hyperion source12,13 manufactured by Oregon Physics (Hillsboro, OR) was used as new oxygen primary negative ion source for the NanoSIMS. This radio frequency source produces a high density oxygen plasma with low temperature ions. The source is operated at a frequency of 40 MHz and a power of 800 W. Compared to the duoplasmatron 7131
DOI: 10.1021/acs.analchem.6b01153 Anal. Chem. 2016, 88, 7130−7136
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Analytical Chemistry
established protocol15 with some modifications in an Automatic Freeze Substitution System AFS2 (Leica Microsystems). The frozen samples were put in liquid nitrogen into cryovials containing 1% osmium tetroxide and 0.1% uranyl acetate and 0.25% glutaraldehyde in anhydrous acetone. The vials were put in AFS2 at −90 °C for 3 days and then warmed to −30 °C for 8 h. The vials were then kept at 0 °C for 1 h and washed with water-free acetone. Sample vials were warmed to room temperature, and the samples were embedded in epoxy resin (EPON 812, Delta Microscopies, Mauressac, France) by infiltrating the samples in a graded series of resin and acetone. The following steps were applied for infiltration: 3:1 acetone/ resin, 1:1 acetone/resin, 1:3 acetone/resin, and 2 baths with 100% resin. Finally, resin embedded samples were polymerized for 48 h at 60 °C. The samples were then cut in 300 nm sections using a diamond knife (Diatome, Biel-Bienne, Switzerland) on an ultramicrotome (EM Ultracut-UC7, Leica Microsystems) and placed on silicon wafers (Wafer Solution, Le Bourget du lac, France) for NanoSIMS measurements. Leaf Cells. Biological Material and Growth Conditions. Arabidopsis thaliana ecotype Columbia 0 was provided by the plant biochemistry group of the Martin Luther University Halle-Wittenberg (Germany). The plant material was grown for 5 weeks under short day conditions (8/16 h, 150 μmol m−2 × s−1) on 1/2 MS medium including vitamins (Duchefa, Haarlem, NL) containing 1% sucrose, 0.8% plant agar (Duchefa), pH 5.8. Sample Preparation for NanoSIMS Analysis. Sample preparation was conducted at the Biozentrum of the University Halle-Wittenberg (Germany). Leaf discs of 2 mm diameter were prepared with a biopsy punch, vacuum-infiltrated with 8% methanol, transferred in aluminum mounts filled with hexadecene, and high-pressure frozen with an HPM 010 (BAL-TEC, Liechtenstein). Subsequently the material was cryo-substituted in 0.25% glutaraldehyde (Sigma, Taufkirchen, Germany) and 0.1% uranyl acetate (Chemapol, Czech Republic) in acetone for 2 days at −80 °C using cryosubstitution equipment (FSU 010, BAL-TEC, Liechtenstein). After an increase of the temperature to −20 °C, the samples were washed 3 times with acetone. This was followed by a stepwise infiltration with embedding medium HM20 (Polysciences Europe, Eppelheim Germany) at −20 °C. Finally, the samples were embedded and polymerized using an UVlamp (24 h; −20 °C). The material was sectioned with an Ultracut S ultramicrotome (Leica, Wetzlar, Germany), and the sections (300 nm) were transferred to silica wafer Plano, Wetzlar, Germany) and air-dried overnight.
source. Prior to the installation of the rf plasma source, experiments were conducted using the standard duoplasmatron oxygen primary ion source. The NanoSIMS instrument is equipped with seven parallel electron multiplier detectors (EM). The mass resolution was adjusted in order to resolve major interferences involving the chosen elements, e.g., MRP (mass resolving power) of 5000 to resolve 40Ca16O from 56Fe. Image processing was performed using ImageJ software (Wayne Rasband, National Institutes of Health (NIH), Bethesda, MD) in addition with the openMIMS plugin developed at the National Resource for Imaging Mass Spectrometry (NRIMS, Cambridge, MA). Chemicals and Materials. All chemicals and reagents were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France and Taufkirchen, Germany) unless stated otherwise. All dilutions and preparations were made with water (18.2 MΩ × cm) obtained from a Milli-Q system (Millipore, Bedford, MA) unless stated otherwise. Reference Materials. Five reference materials (metal alloys) were used to compare the count rates of different elements. ERM-EB317 for aluminum, CRM-296-1 for iron, ERM-EB375 for copper, ERM-EB101a for lead was obtained from the Bundesanstalt für Materialforschung und -prüfung (BAM, Berlin, Germany), and BCR-352 for zinc was obtained from the Bureau of Analyzed Stamples Ltd. (BAS, Middlesbrough, England) . The 1 mm × 1 mm pieces of each standard were taken out and cast into a single sample of 1 cm diameter using Field’s metal, an indium−bismuth−tin alloy (Mindsets, Waltham Cross, England). The sample was then polished with SiC paper (Escil, Chassieu, France) of decreasing grain size, finished using 0.25 μm diamond suspension (Escil, Chassieu, France), and was finally washed using ultrapure ethanol. Sample Preparation. Silicon Grains. Si grains were pressed onto an aluminum matrix and polished to a very high degree. The resulting sharp edges of Si grains are well suited for the purpose of determining the diameter of the primary ion beam using the customary 16−84% criterion. Algae Cells. Biological Material and Growth Conditions. Chlamydomonas reinhardtii wild type strain (11/32b) was obtained from the cell physiology group, Institute of Biology, Plant Physiology, of the University Halle-Wittenberg (Germany). Microalgae were grown in tris-acetate-phosphate (TAP) medium,14 at 22 °C under constant illumination and constant agitation (120 rpm). Fresh TAP medium was inoculated with stock culture in the end of exponential phase to reach an initial optical density (730 nm) of 0.040. To avoid metal contamination, all the glassware was washed with 5% HNO3 before using. Moreover, all the solutions were prepared with ultrapure Milli-Q water. Sample Preparation for NanoSIMS Analysis. Sample preparation was conducted at the Bordeaux Imaging Center (Bordeaux, France). Chemicals for sample preparation were obtained from Delta microscopies (Mauressac, France). In preparation for high-pressure freezing, algae were incubated during 1 h with 150 mM mannitol, and after centrifugation the pellet was included with agarose 2%. Small pieces (200 μm thick and 1.5 mm in length) were cut and were submerged in extracellular cryo-protectant 1-hexadecene. The pieces were placed between two sample holders that formed a chamber (Leica Microsystems, Vienna, Austria). High-pressure freezing was performed immediately (EM HPM 100, Leica Microsystems), and samples were transferred under liquid nitrogen to cryovials. Freeze-substitution was performed following a well-
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RESULTS AND DISCUSSION Characteristics of the Oxygen rf Plasma Ion Source. Using the Wien filter located just underneath the source, the distribution of the ions coming from the source was evaluated. The Wien filter is formed by superimposed electrostatic and magnetic fields that act as a straight-line mass filter by changing the voltage of the electrostatic plates so that only the ionic species of interest and can pass through a diaphragm (D0-3) located on the source axis. The primary beam was then demagnified using an electrostatic lens (L1) and its current measured with a faraday cup (FCP) located at the bottom of the primary column. By scanning the electrostatic field of the Wien filter and leaving the magnetic field unchanged (coil = 2 A), different ions successively passed through the diaphragm revealing the ion distribution. Figure 2 shows that O− was the 7132
DOI: 10.1021/acs.analchem.6b01153 Anal. Chem. 2016, 88, 7130−7136
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Analytical Chemistry
100 nm the rf plasma source provided a current of 2.1 pA which is comparable to the specifications of the cesium source (2 pA).16 In contrast, the duoplasmatron source provided only a current of 0.13 pA. Hence, for a given probe size, the new O− rf plasma source showed similar characteristics as the Cs+ source but provided 16 times more current than the duoplasmatron source. Thus, the higher current density and lower energy distribution provided by the O− rf plasma source allowed the reduction of the probe size in order to achieve a higher lateral resolution. The lowest probe size and thus the highest lateral resolution achieved with the new rf plasma source was 37 nm with a current of 0.15 pA of O− primary ions at an impact energy of 16 keV. Secondary Ion Yield and Apparent Sensitivity. Compared to the duoplasmatron source, the higher current density of the rf plasma source led to a higher atom sputtering rate in the sample and thus to a higher yield in positive secondary ions generation for a given probe size and acquisition time. In order to demonstrate this, both oxygen primary ion sources were compared for the number of counts achievable for five elements (Al, Fe, Cu, Zn, Pb) by analyzing a set of five reference materials. For a correct comparison, the measured count number had to be normalized to the acquisition time, to the probe size, and also to the abundance of the chosen isotope (Figure 4). Thus, all results were expressed in counts per
Figure 2. Determination of the oxygen ion distribution of the primary ion beam using a Wien filter located after the source. O− ions represented approximately 85% of the distribution.
dominant oxygen species (85%) of negative ions produced by the rf plasma source followed by O2− (14%) and O3− and OH− as traces (
