Research Article Received: 2 August 2013
Revised: 28 August 2013
Accepted: 29 August 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 2588–2594 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6726
Laser ablation based bioimaging with simultaneous elemental and molecular mass spectrometry: towards spatially resolved speciation analysis Christina Herdering1, Christoph A. Wehe1, Olga Reifschneider1, Indra Raj2, Giuliano Ciarimboli2, Kurt Diebold3, Christoph Becker4, Michael Sperling1,5 and Uwe Karst1* 1
University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 28/30, 49149 Münster, Germany University of Münster, University Hospital, Experimental Nephrology and Interdisciplinary Center for Clinical Research (IZKF), Albert-Schweizer-Campus 1 – A14, 48149 Münster, Germany 3 Practice of Pathology, Werler Strasse 110, 59063 Hamm, Germany 4 Practice of Oral and Maxillofacial Surgery, Richard-Matthaei-Platz 1, 59065 Hamm, Germany 5 European Virtual Institute for Speciation Analysis (EVISA), Mendelstr. 11, 48149 Münster, Germany 2
RATIONALE: Biological functions of metals are not only specified by the element itself, but also by its chemical form and by its organ, cell and subcellular location. The developed laser ablation based setup enables spatially resolved analysis with simultaneous elemental and molecular mass spectrometry (MS) and promises therefore localization, identification and quantification of metal or heteroelement-containing species in biological samples such as tissue sections. METHODS: A UV laser ablation (LA) system is hyphenated in parallel both with an elemental and a molecular mass spectrometer via flow splitted transfer lines to simultaneously obtain data from both of the mass spectrometers. Elemental MS was performed using inductively coupled plasma (ICP)-MS, whereas atmospheric pressure chemical ionization (APCI)-MS with an orbitrap mass analyzer was utilized for molecular MS. RESULTS: Simultaneous elemental and molecular MS imaging with high lateral resolution down to 25 μm was presented for the staining agents eosin Y and haematoxylin as well as for the chemotherapy drug cisplatin in thin tissue sections. For molecular MS, target compounds were identified by their exact masses and by characteristic fragment ions. CONCLUSIONS: The first simultaneous elemental and molecular MS imaging approach based on laser ablation sampling was introduced for spatially resolved speciation analysis. The combination of the advantages of LA-ICP-MS such as low detection limits and high spatial resolution with information on the chemical identity promises not only localization of metals, but also identification of metal species in biological samples. Therefore, this novel technique opens up new possibilities to address complex challenges in life science research. Copyright © 2013 John Wiley & Sons, Ltd.
Biological functions of metal- or other heteroelementcontaining compounds are typically not dependent on the element itself, but also on its redox state or chemical binding form[1,2] and on its organ, cell and subcellular location. For the examination of the reasons of essential, beneficial or toxic effects of metal species, the information of the total element concentration of bulk (homogenized) samples is not sufficient. Modern speciation analysis began at the end of the 1970s, when Suzuki and Van Loon introduced the coupling of a chromatographic separation technique with an element-specific detection technique, such as atomic spectrometry, for speciesselective detection.[3–5] In the following decades, these so-called hyphenated techniques developed from the determination of
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* Correspondence to: U. Karst, University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 28/30, 49149 Münster, Germany. E-mail: [email protected]
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environmental organometallic pollutants to the characterization of metal-biomolecule interactions in body fluids, cells and tissues.[6,7] Whereas separation and elemental detection methods became more and more selective and sensitive, mainly driven by the development of inductively coupled plasma mass spectrometry (ICP-MS) as the detection system, the identification of unknown species remains challenging as molecular information is completely lost during atomization.[6] To overcome this limitation, molecule-specific detectors such as electrospray ionization (ESI)-MS[8–10] or matrix-assisted laser desorption/ionization (MALDI)-MS[11] were applied for identification of unknown metal species and interacting endogenous molecules. Already by the end of the 1990s, it was recognized that the parallel or even simultaneous detection by elemental and molecular mass spectrometry provides great advantages with respect to complementary information delivered by the two techniques.[12–14] Besides the molecular identity, effects and functions of target species depend on the organ, cell and subcellular location. To examine these distributions, several novel analytical methods
Copyright © 2013 John Wiley & Sons, Ltd.
Bioimaging by simultaneous elemental and molecular mass spectrometry have been developed in the last decades.[15,16] For metal- or heteroelement-containing compounds, laser ablation (LA) coupled to ICP-MS has emerged to become one of the most powerful elemental imaging methods with high spatial resolution down to approximately 2 μm, low detection limits down to sub μg/g and low matrix effects.[17,18] An important application of LA-ICP-MS is the examination of the effects of the chemotherapy drug cisplatin.[19–21] Zoriy et al.[20] and Moreno-Gordalize et al.[21] observed the distribution of platinum in kidneys to study cisplatin-induced nephrotoxicity. Whereas the quantitative sampling by LA and the speciesindependent response by ICP-MS offer several advantages, information on the molecular identity of the metal species and the interacting organic molecules are completely lost upon atomization in the very hot ionization source. While there are powerful molecular imaging methods available such as MALDI-MS imaging[22,23] or secondary ion MS imaging,[22] their parallel use with LA-ICP-MS is not possible and different sample preparation strategies are required. Shelley et al. and Herdering et al. were the first to combine a common LA system as used for LA-ICP-MS with a molecular mass spectrometer for molecular MS imaging.[24,25] Whereas Shelley et al. used a flowing atmospheric pressure afterglow for post-ionization,[24] Herdering et al. applied an only slightly modified APCI source.[25] In this work, a novel approach for simultaneous elemental and molecular bioimaging is presented. The experimental setup is based on a common LA system with a 213 nm laser coupled in parallel both with an elemental mass spectrometer using an ICP as ionization source and with a molecular mass spectrometer with an APCI source. The simultaneous elemental and molecular MS detection is demonstrated for the histological staining agent eosin Y in tissue sections. Furthermore, elemental and molecular bioimaging with high lateral resolution down to 25 μm is presented for the staining agents eosin Y and haematoxylin as well as for the chemotherapy drug cisplatin.
A schematic diagram of the LA-ICP-MS/APCI-MS setup is given in Fig. 1. In detail, spatially resolved sampling was performed with the laser ablation system LSX 213 with a frequency quintupled, Q-switched Nd:YAG laser (λ = 213 nm) from CETAC Technologies (Omaha, NE, USA). As elemental mass spectrometer, an ICP quadrupole mass spectrometer (iCAP Qc, Thermo Fisher Scientific, Bremen, Germany) was used. For molecular MS, an orbitrap mass spectrometer (Exactive Classic HCD, Thermo Fisher Scientific) was used as the determination of the chemical structures in complex biological samples requires high-resolution mass data. As APCI source, the Ion Max source housing of the Exactive Classic HCD was equipped with an APCI probe, a corona needle and a home-built inlet that enables introduction of gaseous samples instead of liquids in the standard mode.[25,26] The parallel coupling of the LA system both with the ICP-MS and the APCI-MS setups was established via flow splitted transfer lines, whereby polyamide tubing with an i.d. of 1 mm was used to minimize the diffusion with acceptable back pressure. With regard to the sensitivity of ICP-MS and APCI-MS, the tubing length was adjusted to a total length of 0.5 m from the ablation cell to the APCI source and to a length of 2.5 m from the ablation cell to the ICP-MS setup so that the larger part of the ablated material was transferred to the less sensitive APCI-MS system. Optical images were recorded using an inverted fluorescence phase-contrast digital microscope BZ-9000E (Keyence Deutschland, Neu-Isenburg, Germany) with CFI Plan Apo 2xλ and CFI Plan Apo 10xλ (Nikon, Düsseldorf, Germany) as lenses. The kidney sections were prepared with a SM 2000R microtome (Leica, Wetzlar, Germany) whereas the lymph nodes were sectioned with an HM 340 E microtome (Thermo Fisher Scientific, Walldorf, Germany).
LA-ICP-MS/APCI-MS of eosin Y stained kidney sections
EXPERIMENTAL Chemicals and consumables
To study the simultaneous elemental and molecular MS detection by LA-ICP-MS/APCI-MS, an eosin Y stained kidney section of a mouse treated with cisplatin was analyzed. FVB/N HanHsd mice (Harlan Laboratories, An Venray, The Netherlands) were treated with 4 mg/kg cisplatin interperitonally (i.p.) twice a week for 4 weeks in the animal facilities of the University Hospital Münster. The experiments were approved by a governmental committee on animal welfare (LaNUV, permission number A 140.2011) and performed in accordance with national animal protection guidelines. Seven weeks after the last cisplatin administration,
Figure 1. Schematic of the LA-ICP-MS/APCI-MS setup.
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Nitric acid (suprapure, 65%) and yttrium and thallium ICP standards (1000 μg/mL) were purchased from Merck (Darmstadt, Germany). Scandium ICP standard (1000 μg/mL) was obtained from SCP Science (Countaboeuf, France). Paraplast X-TRA (melting point 52 °C) and Surgipath Paraplast (melting point 56 °C) were purchased from Leica Biosystems (Wetzlar, Germany). Roti-Histofix 4% (4% formaldehyde in phosphate buffer, acid-free (pH 7)) and eosin Y solution (0.5% in water) were obtained from Carl Roth (Karlsruhe, Germany). Superfrost Plus adhesive microscope slides were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Isobutanol was obtained from Amresco (Solon, OH, USA) and ethanol from AppliChem (Darmstadt, Germany). For lymph node tissue section preparation, formaldehyde was purchased from H. Möller (Steinfurt, Germany), ethanol and xylene from Kassner & Sasse (Dortmund, Germany), haematoxylin solution from Waldeck (Münster, Germany) and Certistain eosin Y C.I. from Merck. Water was purified through an Aquatron Water Stills purification system model A4000D (Barloworld Scientific, Nemours Cedex, France).
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Instrumentation
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the kidney was removed after perfusion through the heart with ice-cold saline followed by 4% paraformaldehyde (Roti-Histofix). Then, the kidney was post-fixed with 4% formaldehyde for 8 h, dehydrated by a sequence of increasing ethanol in water mixtures at room temperature (50% ethanol, 30 min; 70% ethanol, 30 min; 85% ethanol, 30 min; 96% ethanol, 30 min; 100% ethanol, 30 min (twice)) and embedded in paraffin. For embedding, first ethanol was stepwise substituted by isopropanol (Step 1: 25% isopropanol in ethanol 45 min at 60–65 °C; Step 2: 75% isopropanol in ethanol 60 min at 60–65 °C; Step 3: 100% isopropanol overnight at room temperature). The paraffin embedding was performed in five steps: 1. 25% Paraplast X-TRA in isopropanol for 60 min at 60–65 °C; 2. 75% Paraplast X-TRA in isopropanol for 60 min at 60–65 °C; 3. 100% Paraplast X-TRA (melting point 51–53 °C) for 45 min at 60–65 °C; 4. 50% Paraplast X-TRA and 50% Surgipath for 45 min at 60–65 °C; 5. 100% Surgipath for 45 min at 60–65 °C. Tissue sections of 3 μm thickness were obtained with the Leica SM 2000R microtome and mounted onto slides (Super Frost Plus). For tissue dewaxing, the sections were treated with xylene (twice, 5 min) and hydrated with ethanol water mixtures with increasing water percentage (1. 100% ethanol, 2 min; 2. 96% ethanol, 2 min; 3. 70% ethanol, 2 min; 4. 100% water, 2 min). Then, the kidney sections were stained with eosin Y (0.5% eosin Y in water; 3 min), washed twice with tap water and dehydrated with ethanol/water mixtures with increasing ethanol percentage (1. 70% ethanol, 2 min; 2. 95% ethanol, 2 min; 3. 100% ethanol, 2 min). Microphotographs were taken from the stained sections. For LA-ICP-MS/APCI-MS, the stained kidney section was ablated with a laser spot size of 25 μm, a scan rate of 12.5 μm/s, a shot repetition rate of 20 Hz and an averaged output energy of 1.95 mJ/shot, whereby the spatially resolved sampling was performed by subsequent line scans with no gap between the lines. To transfer the ablated sample material to the mass spectrometer, argon was used as carrier gas. For LA-APCI-MS, nitrogen is normally utilized as carrier gas,[25] whereas argon or argon/helium mixtures are used for LA-ICP-MS.[19] As nitrogen has more degrees of freedom than argon, resulting in higher heat capacity, it would destabilize the ICP, whereas helium and argon may cause problems for the vacuum system of the APCI-MS system as they have smaller mean free paths than nitrogen. It was possible to reach a sufficient vacuum by adding a nitrogen flow of 5 a.u. as auxiliary gas to the APCI source. For elemental MS, the ICP-MS instrument was set to a radiofrequency (RF) power of 1550 W, an auxiliary gas flow of 0.8 L/min and a nebulizer gas flow of 0.72 L/min. To stabilize the argon plasma and to detect potential intensity drifts, yttrium and thallium ICP standards (5 μg/mL in 2% nitric acid) were added with an uptake rate of 300 μL/min using a quartz cyclonic spray chamber at 2.7 °C, equipped with a PFA microflow nebulizer as wet aerosol. Furthermore, a 1.8 mm quartz injector and a nickel sampler and skimmer with a 2.8 mm insert were used. To remove polyatomic isobaric interferences, kinetic energy discrimination (KED) with a bias potential of 3 V between cell and quadrupole mass analyzer and a cell gas flow of 5.0 mL/min (8% H2 in He) was used. 79,81Br (dwell time 0.25 s), 89Y (dwell time 0.1 s), 194,195,196 Pt (dwell time 0.1 s) and 203,205Tl (dwell time 0.05 s) were detected with the indicated dwell times and one channel for each isotope. The detected intensities of 89Y and 203,205Tl
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stayed constant over the full analysis time (relative standard deviations of less than 5%) and therefore no drift correction was necessary. For molecular MS, post-ionization was performed by APCI in the positive ion mode with a discharge current of 4 μA and a vaporizer temperature of 300 °C. The orbitrap system was operated in full scan mode in the m/z range 300–700 with a resolving power of 50.000 at m/z 200. The AGC target was set to 5 × 105 and the maximum inject time to 1000 ms. The ion optics settings were optimized for eosin Y and were left unchanged during consecutive experiments (capillary voltage 32.5 V, capillary temperature 250°C, tube lens voltage 120 V, skimmer voltage 34 V). LA-ICP-MS/APCI-MS of haematoxylin and eosin Y (H&E)stained tissue sections Elemental and molecular imaging was demonstrated additionally by analyzing H&E-stained human lymph node sections. The samples were obtained from anonymized human patients as a waste product after surgeries. After removal, the sample preparation was performed according to the following standard protocols for tissue fixation, paraffin embedding and H&E staining in the practice of pathology (Hamm, Germany). First, the lymph nodes were post-fixed with 5% formaldehyde for 48 h at room temperature and dehydrated by a series of ethanol solutions. Before embedding in paraffin, tissues were treated with xylene. The embedded samples were cut as tissue sections of 8 μm thickness. The sections were dewaxed by washing with xylene and a series of graded alcohols. The staining was performed using at first haematoxylin followed by eosin Y. Microphotographs were taken from the stained sections. LA-ICP-MS/APCI-MS was performed as described above with the following changes. The scan rate of the ablation process was increased to 25 μm/s. For elemental MS, scandium and yttrium ICP standards (5 μg/mL in 2% nitric acid) were added and a flow of 5.9 mL/min of the cell gas was used
Figure 2. Chemical formulas of eosin Y (a) and cisplatin (b).
Figure 3. Molecular mass spectrum obtained by LA-APCIMS of an eosin Y stained kidney section of a cisplatintreated mouse.
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Bioimaging by simultaneous elemental and molecular mass spectrometry Table 1. Fragment ions of eosin Y observed by LA-APCI-MS of an eosin Y stained kidney section of a mouse
Formula of detected fragments C20H8O5Br4 + H C20H8O4Br4 + H C20H9O5Br3 + H C20H9O4Br3 + H C19H7O3Br3 + H C20H10O5Br2 + H C20H10O4Br2 + H C19H8O3Br2 + H C20H11O5Br + H C20H11O4Br + H C19H9O3Br + H C20H11O5Br + H C20H11O4Br + H
Calculated mass
Measured mass
δ(Δm/z) [ppm]
Leaving group
648.7137 632.7188 568.8052 552.8103 522.7998 490.8947 474.8998 444.8892 410.9863 394.9913 364.9808 333.0757 317.0808
648.7160 632.7205 568.8073 552.8123 522.8016 490.8964 474.9014 444.8905 410.9876 394.9925 364.9820 333.0769 317.0813
3.5 2.6 3.7 3.6 3.4 3.5 3.4 2.9 3.2 3.0 3.3 3.6 1.6
(Eosin Y) –O – Br + H – Br + H – O – Br + H – CH2O2 – 2Br + 2H – 2Br + 2H – O – 2Br + 2H – CH2O2 – 3Br + 3H – 3Br + 3H – O – 3Br + 3H – CH2O2 – 4Br + 4H – 4Br + 4H – O
for the KED mode. The recorded isotopes were 27Al (dwell time 0.4 s), 79,81Br (dwell time 0.2 s), 45Sc (dwell time 0.1 s) and 89Y (dwell time 0.1 s). For molecular MS, the scan range of the orbitrap system was expanded to m/z 100–1000. Data processing Data processing was performed using Origin 8.5.0 (OriginLab Corporations, Northampton, MS, USA). The presented mass spectra were background corrected using the mass spectrum of a microscope slide as background. The spatially resolved images of the tissue section were finally obtained by conversion of the transient signals of the extracted ion traces into color-coded 2D distribution images.
RESULTS Simultaneous elemental and molecular MS
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Figure 4. Ion traces obtained by LA-ICP-MS/APCI-MS of a selected line of an eosin Y stained kidney of a cisplatin-treated mouse: (a) APCI-MS: [eosin Y + H]+, m/z 648.7037-648.7237; (b) APCI-MS: [eosin Y–Br + 2H]+, m/z 568.8052 ± 0.01; (c) ICP-MS: 81Br; and (d) ICP-MS: 79Br. correlate well with the traces for 79Br and 81Br. Therefore, LA-ICP-MS/APCI-MS is suitable for simultaneous elemental and molecular MS detection. Furthermore, the ion traces of 79 Br and 81Br correspond even in the absolute intensities as expected due to their isotopic ratio. For eosin Y and its fragment ion, the relative intensities are in good accordance. Thus, compounds can be identified and detected by molecular MS by both their [M + H]+ ions and characteristic fragment ions.
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Simultaneous elemental and molecular MS was performed by LA-ICP-MS/APCI-MS of an eosin Y stained kidney section. Eosin Y is an acid, anionic Romanowsky stain that interacts with proteins via ion-exchange processes.[27] The chemical formula of eosin is shown in Fig. 2(a). Due to the bromine, eosin Y can be detected in tissue sections by LA-ICP-MS.[28] Figure 3 shows one representative molecular mass spectrum of the kidney section obtained by LA-APCI-MS. Eosin Y was identified as the [M + H]+ adduct by its exact mass with a deviation between calculated and detected exact masses of 3.5 ppm and by its characteristic isotopic pattern. In addition to the signal group of eosin Y [M + H]+, several fragment ions of eosin Y were identified. These fragments are summarized with their obtained exact masses, the proposed molecular formula and the determined deviations between calculated and detected masses in Table 1. Therefore, eosin Y is detectable as 79Br or 81Br with ICP-MS and as the eosin Y [M + H]+ ion or characteristic fragment ions with APCI-MS. To prove the simultaneous elemental and molecular MS detection, Fig. 4 shows the ion traces for eosin Y [M + H]+ (Fig. 4(a)) and the fragment ion [eosin Y–Br + 2H]+ as well as for 81Br (Fig. 4(c)) and 79Br (Fig. 4(d)) observed by LA-ICPMS/APCI-MS of a selected line of the kidney section. The ion traces for eosin Y [M + H]+ and its fragment ion
C. Herdering et al. Bioimaging of cisplatin and eosin Y Elemental and molecular imaging was performed by LA-ICPMS/APCI-MS of a stained kidney section of a cisplatin-treated mouse. Examination of the reasons for the cisplatin-induced nephrotoxicity by LA-ICP-MS requires high spatial resolution down to 50 μm, low limits of detection down to 10 μg/g platinum and low matrix effects.[20] Figure 5(b) shows the platinum distribution (195Pt) obtained by LA-ICP-MS/APCIMS of a 3 μm thick kidney section of a mouse treated with cisplatin (8 × 4 mg/kg over 8 weeks) and sacrificed 7 weeks after the last cisplatin treatment. The platinum is heterogeneously distributed with an accumulation in the kidney cortex. This part of the kidney contains the proximal tubules, which are specifically damaged by cisplatin.[29] Furthermore, these results are in accordance with the findings of Moreno-Gordaliza et al.[21] As a result, the advantages of LA-ICP-MS including high spatial resolution, high sensitivity, species-independent response and low matrix effects are preserved using the simultaneous LA-ICP-MS/APCI-MS setup. The simultaneous elemental and molecular MS imaging is presented in Fig. 5 for the eosin and bromine distribution, respectively. The obtained images for 79Br (Fig. 5(c)), 81Br (Fig. 5(d)), eosin [M + H]+ (Fig. 5(e)) and the fragment ion [eosinY–Br + 2H]+ correlate well with each other and with the red color of eosin Y in the microphotograph (Fig. 5(a)). In detail, the white or
brighter red regions in the optical image correlate with lower intensities in the ion images. Therefore, LA-ICPMS/APCI-MS is suitable for simultaneous elemental and molecular MS imaging of biological samples including thin tissue sections with high spatial resolution down to 25 μm. For molecular MS, compounds are detected as [M + H]+ ions and characteristic fragment ions, respectively. As a result, LA-ICP-MS/APCI-MS is suitable for spatially resolved speciation analysis. The advantages of LA-ICP-MS, including high spatial resolution, high sensitivity, speciesindependent response and low matrix effects, are combined with information on the chemical structure. Using a highresolution mass analyzer for molecular MS, it is possible to handle the complex biological sample matrices and to identify target species by their exact masses and characteristic fragment ions. Imaging of haematoxylin and eosin Y Simultaneous elemental and molecular imaging of a biological sample was further performed by LA-ICP-MS/ APCI-MS of a haematoxylin and eosin Y (H&E)-stained human lymph node. H&E staining is a standard procedure in histology to improve the microscopic visualization of the morphology of a tissue section.[30] Haematoxylin stains the cell nuclei in deep blue, whereas eosin Y dyes cytoplasmic materials in red.[30] Like eosin Y, haematoxylin is detectable by ICP-MS due to its heteroelement aluminum.[28] Figure 6(a)
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Figure 5. Microphotograph (a) and ion images (b–f) of an eosin Y stained kidney section of a cisplatin-treated mouse. Ion images were obtained by LA-ICP-MS/APCI-MS with a spatial resolution of 25 μm for 195Pt (b), 79Br (c) and 81Br (d) by ICP-MS as well as for eosin Y [M + H]+ m/z 648.7137 ± 0.01 (e) and the fragment ion [eosin Y–Br + 2H]+ m/z 568.8052 ± 0.01 (f)) by APCI-MS.
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Copyright © 2013 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2013, 27, 2588–2594
Bioimaging by simultaneous elemental and molecular mass spectrometry
Figure 6. Microphotograph (a) and ion images (b–d) of a haematoxylin and eosin Y stained human lymph node. Ion images were obtained by LA-ICP-MS/APCI-MS with a spatial resolution of 25 μm for 27Al (b) and 79Br (c) by ICP-MS as well as for eosin Y [M + H]+ m/z 648.7137 ± 0.01 (d) by APCI-MS. shows a microphotograph of the stained lymph node section. The morphological structure of the lymph node is visible, but the blue color of haematoxylin partly covers the red color of the eosin Y. The ion images for 79Br (Fig. 6(c)) and eosin Y [M + H]+ (Fig. 6(d)) clearly reflect the distribution of eosin Y in the lymph node section, despite the presence of haematoxylin. Furthermore, the ion image for 79Br obtained by ICP-MS corresponds to the ion image of eosin Y [M + H]+ obtained by APCI-MS. Therefore, LA-ICP-MS/APCI-MS is suitable for simultaneous elemental and molecular imaging. Besides, Fig. 6 (b) shows the obtained ion image of 27Al. It correlates well with the blue color in the optical image (Fig. 6(a)).
In conclusion, laser ablation coupled in parallel both with elemental and molecular MS is a promising method for the localization and characterization of metal species and their interacting organic molecules in biological samples such as tissue sections. Especially for complex samples and questions in life science research, this novel hyphenation technique opens up new analytical possibilities.
Acknowledgement This work was supported in part by the Cells in Motion Cluster of Excellence (CiM - EXC 1003) Münster, Germany (project FF-2013-17) and by the Interdisciplinary Center for Clinical Research (IZKF) Münster (Grant Cia2/013/13).
DISCUSSION
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The first simultaneous elemental and molecular mass spectrometric imaging approach based on laser ablation sampling has been developed. The presented results demonstrate the benefits of the simultaneous LA-ICP-MS/ APCI-MS setup for spatially resolved speciation analysis. The advantages of LA-ICP-MS including high spatial resolution, low detection limits, species-independent response, large dynamic range and low matrix effects for metal- and heteroelement-containing compounds are combined with information on the chemical identity of the metal species or interacting organic molecules. The simple setup composed of established and mostly commercially available equipment offers flexible ion source and mass analyzer combinations and promises therefore a free choice of the MS instruments depending on target compounds or scientific question.
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[19] O. Reifschneider, C. A. Wehe, I. Raj, J. Ehmcke, G. Ciarimboli, M. Sperling, U. Karst. Quantitative bioimaging of platinum in polymer embedded mouse organs by laser ablation ICP-MS. Metallomics 2013, 5, 1440. [20] M. Zoriy, A. Matusch, T. Spruss, J. S. Becker. Laser ablation inductively coupled plasma mass spectrometry for imaging of copper, zinc, and platinum in thin sections of a kidney from a mouse treated with cis-platin. Int. J. Mass Spectrom. 2007, 260, 102. [21] E. Moreno-Gordaliza, C. Giesen, A. Lazaro, D. EstebanFernandez, B. Humanes, B. Canas, U. Panne, A. Tejedor, N. Jakubowski, M. M. Gomez-Gomez. Elemental bioimaging in kidney by LA-ICP-MS as a tool to study nephrotoxicity and renal protective strategies in cisplatin therapies. Anal. Chem. 2011, 83, 7933. [22] L. A. McDonnell, R. M. A. Heeren. Imaging mass spectrometry. Mass Spectrom. Rev. 2007, 26, 606. [23] A. Römpp, B. Spengler. Mass spectrometry imaging with high resolution in mass and space. Histochem. Cell Biol. 2013, 139, 759. [24] J. T. Shelley, S. J. Ray, G. M. Hieftje. Laser ablation coupled to a flowing atmospheric pressure afterglow for ambient mass spectral imaging. Anal. Chem. 2008, 80, 8308. [25] C. Herdering, O. Reifschneider, C. A. Wehe, M. Sperling, U. Karst. Ambient molecular imaging by laser ablation atmospheric pressure chemical ionization mass spectrometry. Rapid Commun. Mass Spectrom. 2013, 27, 2595. [26] C. Herdering, O. Reifschneider, C. A. Wehe, M. Sperling, U. Karst. U.S. provisional patent application pending, US 61/757, 252, 2013. [27] R. W. Horobin. How Romanowsky stains work and why they remain valuable – including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme. Biotech. Histochem. 2011, 86, 36. [28] O. Reifschneider, C. A. Wehe, K. Diebold, C. Becker, M. Sperling, U. Karst. Elemental bioimaging of haematoxylin and eosin-stained tissues by laser ablation ICP-MS. J. Anal. At. Spectrom. 2013, 28, 989. [29] D. M. Townsend, M. Deng, L. Zhang, M. G. Lapus, M. H. Hanigan. Metabolism of Cisplatin to a nephrotoxin in proximal tubule cells. J. Am. Soc. Nephrol. 2003, 14, 1. [30] R. W. Horobin, J. A. Kiernan. Conn’s Biological Stains: A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine. BIOS Scientific Publ., 2002.
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Revised: 28 August 2013
Accepted: 29 August 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 2588–2594 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6726
Laser ablation based bioimaging with simultaneous elemental and molecular mass spectrometry: towards spatially resolved speciation analysis Christina Herdering1, Christoph A. Wehe1, Olga Reifschneider1, Indra Raj2, Giuliano Ciarimboli2, Kurt Diebold3, Christoph Becker4, Michael Sperling1,5 and Uwe Karst1* 1
University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 28/30, 49149 Münster, Germany University of Münster, University Hospital, Experimental Nephrology and Interdisciplinary Center for Clinical Research (IZKF), Albert-Schweizer-Campus 1 – A14, 48149 Münster, Germany 3 Practice of Pathology, Werler Strasse 110, 59063 Hamm, Germany 4 Practice of Oral and Maxillofacial Surgery, Richard-Matthaei-Platz 1, 59065 Hamm, Germany 5 European Virtual Institute for Speciation Analysis (EVISA), Mendelstr. 11, 48149 Münster, Germany 2
RATIONALE: Biological functions of metals are not only specified by the element itself, but also by its chemical form and by its organ, cell and subcellular location. The developed laser ablation based setup enables spatially resolved analysis with simultaneous elemental and molecular mass spectrometry (MS) and promises therefore localization, identification and quantification of metal or heteroelement-containing species in biological samples such as tissue sections. METHODS: A UV laser ablation (LA) system is hyphenated in parallel both with an elemental and a molecular mass spectrometer via flow splitted transfer lines to simultaneously obtain data from both of the mass spectrometers. Elemental MS was performed using inductively coupled plasma (ICP)-MS, whereas atmospheric pressure chemical ionization (APCI)-MS with an orbitrap mass analyzer was utilized for molecular MS. RESULTS: Simultaneous elemental and molecular MS imaging with high lateral resolution down to 25 μm was presented for the staining agents eosin Y and haematoxylin as well as for the chemotherapy drug cisplatin in thin tissue sections. For molecular MS, target compounds were identified by their exact masses and by characteristic fragment ions. CONCLUSIONS: The first simultaneous elemental and molecular MS imaging approach based on laser ablation sampling was introduced for spatially resolved speciation analysis. The combination of the advantages of LA-ICP-MS such as low detection limits and high spatial resolution with information on the chemical identity promises not only localization of metals, but also identification of metal species in biological samples. Therefore, this novel technique opens up new possibilities to address complex challenges in life science research. Copyright © 2013 John Wiley & Sons, Ltd.
Biological functions of metal- or other heteroelementcontaining compounds are typically not dependent on the element itself, but also on its redox state or chemical binding form[1,2] and on its organ, cell and subcellular location. For the examination of the reasons of essential, beneficial or toxic effects of metal species, the information of the total element concentration of bulk (homogenized) samples is not sufficient. Modern speciation analysis began at the end of the 1970s, when Suzuki and Van Loon introduced the coupling of a chromatographic separation technique with an element-specific detection technique, such as atomic spectrometry, for speciesselective detection.[3–5] In the following decades, these so-called hyphenated techniques developed from the determination of
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* Correspondence to: U. Karst, University of Münster, Institute of Inorganic and Analytical Chemistry, Corrensstr. 28/30, 49149 Münster, Germany. E-mail: [email protected]
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environmental organometallic pollutants to the characterization of metal-biomolecule interactions in body fluids, cells and tissues.[6,7] Whereas separation and elemental detection methods became more and more selective and sensitive, mainly driven by the development of inductively coupled plasma mass spectrometry (ICP-MS) as the detection system, the identification of unknown species remains challenging as molecular information is completely lost during atomization.[6] To overcome this limitation, molecule-specific detectors such as electrospray ionization (ESI)-MS[8–10] or matrix-assisted laser desorption/ionization (MALDI)-MS[11] were applied for identification of unknown metal species and interacting endogenous molecules. Already by the end of the 1990s, it was recognized that the parallel or even simultaneous detection by elemental and molecular mass spectrometry provides great advantages with respect to complementary information delivered by the two techniques.[12–14] Besides the molecular identity, effects and functions of target species depend on the organ, cell and subcellular location. To examine these distributions, several novel analytical methods
Copyright © 2013 John Wiley & Sons, Ltd.
Bioimaging by simultaneous elemental and molecular mass spectrometry have been developed in the last decades.[15,16] For metal- or heteroelement-containing compounds, laser ablation (LA) coupled to ICP-MS has emerged to become one of the most powerful elemental imaging methods with high spatial resolution down to approximately 2 μm, low detection limits down to sub μg/g and low matrix effects.[17,18] An important application of LA-ICP-MS is the examination of the effects of the chemotherapy drug cisplatin.[19–21] Zoriy et al.[20] and Moreno-Gordalize et al.[21] observed the distribution of platinum in kidneys to study cisplatin-induced nephrotoxicity. Whereas the quantitative sampling by LA and the speciesindependent response by ICP-MS offer several advantages, information on the molecular identity of the metal species and the interacting organic molecules are completely lost upon atomization in the very hot ionization source. While there are powerful molecular imaging methods available such as MALDI-MS imaging[22,23] or secondary ion MS imaging,[22] their parallel use with LA-ICP-MS is not possible and different sample preparation strategies are required. Shelley et al. and Herdering et al. were the first to combine a common LA system as used for LA-ICP-MS with a molecular mass spectrometer for molecular MS imaging.[24,25] Whereas Shelley et al. used a flowing atmospheric pressure afterglow for post-ionization,[24] Herdering et al. applied an only slightly modified APCI source.[25] In this work, a novel approach for simultaneous elemental and molecular bioimaging is presented. The experimental setup is based on a common LA system with a 213 nm laser coupled in parallel both with an elemental mass spectrometer using an ICP as ionization source and with a molecular mass spectrometer with an APCI source. The simultaneous elemental and molecular MS detection is demonstrated for the histological staining agent eosin Y in tissue sections. Furthermore, elemental and molecular bioimaging with high lateral resolution down to 25 μm is presented for the staining agents eosin Y and haematoxylin as well as for the chemotherapy drug cisplatin.
A schematic diagram of the LA-ICP-MS/APCI-MS setup is given in Fig. 1. In detail, spatially resolved sampling was performed with the laser ablation system LSX 213 with a frequency quintupled, Q-switched Nd:YAG laser (λ = 213 nm) from CETAC Technologies (Omaha, NE, USA). As elemental mass spectrometer, an ICP quadrupole mass spectrometer (iCAP Qc, Thermo Fisher Scientific, Bremen, Germany) was used. For molecular MS, an orbitrap mass spectrometer (Exactive Classic HCD, Thermo Fisher Scientific) was used as the determination of the chemical structures in complex biological samples requires high-resolution mass data. As APCI source, the Ion Max source housing of the Exactive Classic HCD was equipped with an APCI probe, a corona needle and a home-built inlet that enables introduction of gaseous samples instead of liquids in the standard mode.[25,26] The parallel coupling of the LA system both with the ICP-MS and the APCI-MS setups was established via flow splitted transfer lines, whereby polyamide tubing with an i.d. of 1 mm was used to minimize the diffusion with acceptable back pressure. With regard to the sensitivity of ICP-MS and APCI-MS, the tubing length was adjusted to a total length of 0.5 m from the ablation cell to the APCI source and to a length of 2.5 m from the ablation cell to the ICP-MS setup so that the larger part of the ablated material was transferred to the less sensitive APCI-MS system. Optical images were recorded using an inverted fluorescence phase-contrast digital microscope BZ-9000E (Keyence Deutschland, Neu-Isenburg, Germany) with CFI Plan Apo 2xλ and CFI Plan Apo 10xλ (Nikon, Düsseldorf, Germany) as lenses. The kidney sections were prepared with a SM 2000R microtome (Leica, Wetzlar, Germany) whereas the lymph nodes were sectioned with an HM 340 E microtome (Thermo Fisher Scientific, Walldorf, Germany).
LA-ICP-MS/APCI-MS of eosin Y stained kidney sections
EXPERIMENTAL Chemicals and consumables
To study the simultaneous elemental and molecular MS detection by LA-ICP-MS/APCI-MS, an eosin Y stained kidney section of a mouse treated with cisplatin was analyzed. FVB/N HanHsd mice (Harlan Laboratories, An Venray, The Netherlands) were treated with 4 mg/kg cisplatin interperitonally (i.p.) twice a week for 4 weeks in the animal facilities of the University Hospital Münster. The experiments were approved by a governmental committee on animal welfare (LaNUV, permission number A 140.2011) and performed in accordance with national animal protection guidelines. Seven weeks after the last cisplatin administration,
Figure 1. Schematic of the LA-ICP-MS/APCI-MS setup.
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Nitric acid (suprapure, 65%) and yttrium and thallium ICP standards (1000 μg/mL) were purchased from Merck (Darmstadt, Germany). Scandium ICP standard (1000 μg/mL) was obtained from SCP Science (Countaboeuf, France). Paraplast X-TRA (melting point 52 °C) and Surgipath Paraplast (melting point 56 °C) were purchased from Leica Biosystems (Wetzlar, Germany). Roti-Histofix 4% (4% formaldehyde in phosphate buffer, acid-free (pH 7)) and eosin Y solution (0.5% in water) were obtained from Carl Roth (Karlsruhe, Germany). Superfrost Plus adhesive microscope slides were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Isobutanol was obtained from Amresco (Solon, OH, USA) and ethanol from AppliChem (Darmstadt, Germany). For lymph node tissue section preparation, formaldehyde was purchased from H. Möller (Steinfurt, Germany), ethanol and xylene from Kassner & Sasse (Dortmund, Germany), haematoxylin solution from Waldeck (Münster, Germany) and Certistain eosin Y C.I. from Merck. Water was purified through an Aquatron Water Stills purification system model A4000D (Barloworld Scientific, Nemours Cedex, France).
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Instrumentation
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the kidney was removed after perfusion through the heart with ice-cold saline followed by 4% paraformaldehyde (Roti-Histofix). Then, the kidney was post-fixed with 4% formaldehyde for 8 h, dehydrated by a sequence of increasing ethanol in water mixtures at room temperature (50% ethanol, 30 min; 70% ethanol, 30 min; 85% ethanol, 30 min; 96% ethanol, 30 min; 100% ethanol, 30 min (twice)) and embedded in paraffin. For embedding, first ethanol was stepwise substituted by isopropanol (Step 1: 25% isopropanol in ethanol 45 min at 60–65 °C; Step 2: 75% isopropanol in ethanol 60 min at 60–65 °C; Step 3: 100% isopropanol overnight at room temperature). The paraffin embedding was performed in five steps: 1. 25% Paraplast X-TRA in isopropanol for 60 min at 60–65 °C; 2. 75% Paraplast X-TRA in isopropanol for 60 min at 60–65 °C; 3. 100% Paraplast X-TRA (melting point 51–53 °C) for 45 min at 60–65 °C; 4. 50% Paraplast X-TRA and 50% Surgipath for 45 min at 60–65 °C; 5. 100% Surgipath for 45 min at 60–65 °C. Tissue sections of 3 μm thickness were obtained with the Leica SM 2000R microtome and mounted onto slides (Super Frost Plus). For tissue dewaxing, the sections were treated with xylene (twice, 5 min) and hydrated with ethanol water mixtures with increasing water percentage (1. 100% ethanol, 2 min; 2. 96% ethanol, 2 min; 3. 70% ethanol, 2 min; 4. 100% water, 2 min). Then, the kidney sections were stained with eosin Y (0.5% eosin Y in water; 3 min), washed twice with tap water and dehydrated with ethanol/water mixtures with increasing ethanol percentage (1. 70% ethanol, 2 min; 2. 95% ethanol, 2 min; 3. 100% ethanol, 2 min). Microphotographs were taken from the stained sections. For LA-ICP-MS/APCI-MS, the stained kidney section was ablated with a laser spot size of 25 μm, a scan rate of 12.5 μm/s, a shot repetition rate of 20 Hz and an averaged output energy of 1.95 mJ/shot, whereby the spatially resolved sampling was performed by subsequent line scans with no gap between the lines. To transfer the ablated sample material to the mass spectrometer, argon was used as carrier gas. For LA-APCI-MS, nitrogen is normally utilized as carrier gas,[25] whereas argon or argon/helium mixtures are used for LA-ICP-MS.[19] As nitrogen has more degrees of freedom than argon, resulting in higher heat capacity, it would destabilize the ICP, whereas helium and argon may cause problems for the vacuum system of the APCI-MS system as they have smaller mean free paths than nitrogen. It was possible to reach a sufficient vacuum by adding a nitrogen flow of 5 a.u. as auxiliary gas to the APCI source. For elemental MS, the ICP-MS instrument was set to a radiofrequency (RF) power of 1550 W, an auxiliary gas flow of 0.8 L/min and a nebulizer gas flow of 0.72 L/min. To stabilize the argon plasma and to detect potential intensity drifts, yttrium and thallium ICP standards (5 μg/mL in 2% nitric acid) were added with an uptake rate of 300 μL/min using a quartz cyclonic spray chamber at 2.7 °C, equipped with a PFA microflow nebulizer as wet aerosol. Furthermore, a 1.8 mm quartz injector and a nickel sampler and skimmer with a 2.8 mm insert were used. To remove polyatomic isobaric interferences, kinetic energy discrimination (KED) with a bias potential of 3 V between cell and quadrupole mass analyzer and a cell gas flow of 5.0 mL/min (8% H2 in He) was used. 79,81Br (dwell time 0.25 s), 89Y (dwell time 0.1 s), 194,195,196 Pt (dwell time 0.1 s) and 203,205Tl (dwell time 0.05 s) were detected with the indicated dwell times and one channel for each isotope. The detected intensities of 89Y and 203,205Tl
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stayed constant over the full analysis time (relative standard deviations of less than 5%) and therefore no drift correction was necessary. For molecular MS, post-ionization was performed by APCI in the positive ion mode with a discharge current of 4 μA and a vaporizer temperature of 300 °C. The orbitrap system was operated in full scan mode in the m/z range 300–700 with a resolving power of 50.000 at m/z 200. The AGC target was set to 5 × 105 and the maximum inject time to 1000 ms. The ion optics settings were optimized for eosin Y and were left unchanged during consecutive experiments (capillary voltage 32.5 V, capillary temperature 250°C, tube lens voltage 120 V, skimmer voltage 34 V). LA-ICP-MS/APCI-MS of haematoxylin and eosin Y (H&E)stained tissue sections Elemental and molecular imaging was demonstrated additionally by analyzing H&E-stained human lymph node sections. The samples were obtained from anonymized human patients as a waste product after surgeries. After removal, the sample preparation was performed according to the following standard protocols for tissue fixation, paraffin embedding and H&E staining in the practice of pathology (Hamm, Germany). First, the lymph nodes were post-fixed with 5% formaldehyde for 48 h at room temperature and dehydrated by a series of ethanol solutions. Before embedding in paraffin, tissues were treated with xylene. The embedded samples were cut as tissue sections of 8 μm thickness. The sections were dewaxed by washing with xylene and a series of graded alcohols. The staining was performed using at first haematoxylin followed by eosin Y. Microphotographs were taken from the stained sections. LA-ICP-MS/APCI-MS was performed as described above with the following changes. The scan rate of the ablation process was increased to 25 μm/s. For elemental MS, scandium and yttrium ICP standards (5 μg/mL in 2% nitric acid) were added and a flow of 5.9 mL/min of the cell gas was used
Figure 2. Chemical formulas of eosin Y (a) and cisplatin (b).
Figure 3. Molecular mass spectrum obtained by LA-APCIMS of an eosin Y stained kidney section of a cisplatintreated mouse.
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Bioimaging by simultaneous elemental and molecular mass spectrometry Table 1. Fragment ions of eosin Y observed by LA-APCI-MS of an eosin Y stained kidney section of a mouse
Formula of detected fragments C20H8O5Br4 + H C20H8O4Br4 + H C20H9O5Br3 + H C20H9O4Br3 + H C19H7O3Br3 + H C20H10O5Br2 + H C20H10O4Br2 + H C19H8O3Br2 + H C20H11O5Br + H C20H11O4Br + H C19H9O3Br + H C20H11O5Br + H C20H11O4Br + H
Calculated mass
Measured mass
δ(Δm/z) [ppm]
Leaving group
648.7137 632.7188 568.8052 552.8103 522.7998 490.8947 474.8998 444.8892 410.9863 394.9913 364.9808 333.0757 317.0808
648.7160 632.7205 568.8073 552.8123 522.8016 490.8964 474.9014 444.8905 410.9876 394.9925 364.9820 333.0769 317.0813
3.5 2.6 3.7 3.6 3.4 3.5 3.4 2.9 3.2 3.0 3.3 3.6 1.6
(Eosin Y) –O – Br + H – Br + H – O – Br + H – CH2O2 – 2Br + 2H – 2Br + 2H – O – 2Br + 2H – CH2O2 – 3Br + 3H – 3Br + 3H – O – 3Br + 3H – CH2O2 – 4Br + 4H – 4Br + 4H – O
for the KED mode. The recorded isotopes were 27Al (dwell time 0.4 s), 79,81Br (dwell time 0.2 s), 45Sc (dwell time 0.1 s) and 89Y (dwell time 0.1 s). For molecular MS, the scan range of the orbitrap system was expanded to m/z 100–1000. Data processing Data processing was performed using Origin 8.5.0 (OriginLab Corporations, Northampton, MS, USA). The presented mass spectra were background corrected using the mass spectrum of a microscope slide as background. The spatially resolved images of the tissue section were finally obtained by conversion of the transient signals of the extracted ion traces into color-coded 2D distribution images.
RESULTS Simultaneous elemental and molecular MS
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Figure 4. Ion traces obtained by LA-ICP-MS/APCI-MS of a selected line of an eosin Y stained kidney of a cisplatin-treated mouse: (a) APCI-MS: [eosin Y + H]+, m/z 648.7037-648.7237; (b) APCI-MS: [eosin Y–Br + 2H]+, m/z 568.8052 ± 0.01; (c) ICP-MS: 81Br; and (d) ICP-MS: 79Br. correlate well with the traces for 79Br and 81Br. Therefore, LA-ICP-MS/APCI-MS is suitable for simultaneous elemental and molecular MS detection. Furthermore, the ion traces of 79 Br and 81Br correspond even in the absolute intensities as expected due to their isotopic ratio. For eosin Y and its fragment ion, the relative intensities are in good accordance. Thus, compounds can be identified and detected by molecular MS by both their [M + H]+ ions and characteristic fragment ions.
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Simultaneous elemental and molecular MS was performed by LA-ICP-MS/APCI-MS of an eosin Y stained kidney section. Eosin Y is an acid, anionic Romanowsky stain that interacts with proteins via ion-exchange processes.[27] The chemical formula of eosin is shown in Fig. 2(a). Due to the bromine, eosin Y can be detected in tissue sections by LA-ICP-MS.[28] Figure 3 shows one representative molecular mass spectrum of the kidney section obtained by LA-APCI-MS. Eosin Y was identified as the [M + H]+ adduct by its exact mass with a deviation between calculated and detected exact masses of 3.5 ppm and by its characteristic isotopic pattern. In addition to the signal group of eosin Y [M + H]+, several fragment ions of eosin Y were identified. These fragments are summarized with their obtained exact masses, the proposed molecular formula and the determined deviations between calculated and detected masses in Table 1. Therefore, eosin Y is detectable as 79Br or 81Br with ICP-MS and as the eosin Y [M + H]+ ion or characteristic fragment ions with APCI-MS. To prove the simultaneous elemental and molecular MS detection, Fig. 4 shows the ion traces for eosin Y [M + H]+ (Fig. 4(a)) and the fragment ion [eosin Y–Br + 2H]+ as well as for 81Br (Fig. 4(c)) and 79Br (Fig. 4(d)) observed by LA-ICPMS/APCI-MS of a selected line of the kidney section. The ion traces for eosin Y [M + H]+ and its fragment ion
C. Herdering et al. Bioimaging of cisplatin and eosin Y Elemental and molecular imaging was performed by LA-ICPMS/APCI-MS of a stained kidney section of a cisplatin-treated mouse. Examination of the reasons for the cisplatin-induced nephrotoxicity by LA-ICP-MS requires high spatial resolution down to 50 μm, low limits of detection down to 10 μg/g platinum and low matrix effects.[20] Figure 5(b) shows the platinum distribution (195Pt) obtained by LA-ICP-MS/APCIMS of a 3 μm thick kidney section of a mouse treated with cisplatin (8 × 4 mg/kg over 8 weeks) and sacrificed 7 weeks after the last cisplatin treatment. The platinum is heterogeneously distributed with an accumulation in the kidney cortex. This part of the kidney contains the proximal tubules, which are specifically damaged by cisplatin.[29] Furthermore, these results are in accordance with the findings of Moreno-Gordaliza et al.[21] As a result, the advantages of LA-ICP-MS including high spatial resolution, high sensitivity, species-independent response and low matrix effects are preserved using the simultaneous LA-ICP-MS/APCI-MS setup. The simultaneous elemental and molecular MS imaging is presented in Fig. 5 for the eosin and bromine distribution, respectively. The obtained images for 79Br (Fig. 5(c)), 81Br (Fig. 5(d)), eosin [M + H]+ (Fig. 5(e)) and the fragment ion [eosinY–Br + 2H]+ correlate well with each other and with the red color of eosin Y in the microphotograph (Fig. 5(a)). In detail, the white or
brighter red regions in the optical image correlate with lower intensities in the ion images. Therefore, LA-ICPMS/APCI-MS is suitable for simultaneous elemental and molecular MS imaging of biological samples including thin tissue sections with high spatial resolution down to 25 μm. For molecular MS, compounds are detected as [M + H]+ ions and characteristic fragment ions, respectively. As a result, LA-ICP-MS/APCI-MS is suitable for spatially resolved speciation analysis. The advantages of LA-ICP-MS, including high spatial resolution, high sensitivity, speciesindependent response and low matrix effects, are combined with information on the chemical structure. Using a highresolution mass analyzer for molecular MS, it is possible to handle the complex biological sample matrices and to identify target species by their exact masses and characteristic fragment ions. Imaging of haematoxylin and eosin Y Simultaneous elemental and molecular imaging of a biological sample was further performed by LA-ICP-MS/ APCI-MS of a haematoxylin and eosin Y (H&E)-stained human lymph node. H&E staining is a standard procedure in histology to improve the microscopic visualization of the morphology of a tissue section.[30] Haematoxylin stains the cell nuclei in deep blue, whereas eosin Y dyes cytoplasmic materials in red.[30] Like eosin Y, haematoxylin is detectable by ICP-MS due to its heteroelement aluminum.[28] Figure 6(a)
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Figure 5. Microphotograph (a) and ion images (b–f) of an eosin Y stained kidney section of a cisplatin-treated mouse. Ion images were obtained by LA-ICP-MS/APCI-MS with a spatial resolution of 25 μm for 195Pt (b), 79Br (c) and 81Br (d) by ICP-MS as well as for eosin Y [M + H]+ m/z 648.7137 ± 0.01 (e) and the fragment ion [eosin Y–Br + 2H]+ m/z 568.8052 ± 0.01 (f)) by APCI-MS.
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Bioimaging by simultaneous elemental and molecular mass spectrometry
Figure 6. Microphotograph (a) and ion images (b–d) of a haematoxylin and eosin Y stained human lymph node. Ion images were obtained by LA-ICP-MS/APCI-MS with a spatial resolution of 25 μm for 27Al (b) and 79Br (c) by ICP-MS as well as for eosin Y [M + H]+ m/z 648.7137 ± 0.01 (d) by APCI-MS. shows a microphotograph of the stained lymph node section. The morphological structure of the lymph node is visible, but the blue color of haematoxylin partly covers the red color of the eosin Y. The ion images for 79Br (Fig. 6(c)) and eosin Y [M + H]+ (Fig. 6(d)) clearly reflect the distribution of eosin Y in the lymph node section, despite the presence of haematoxylin. Furthermore, the ion image for 79Br obtained by ICP-MS corresponds to the ion image of eosin Y [M + H]+ obtained by APCI-MS. Therefore, LA-ICP-MS/APCI-MS is suitable for simultaneous elemental and molecular imaging. Besides, Fig. 6 (b) shows the obtained ion image of 27Al. It correlates well with the blue color in the optical image (Fig. 6(a)).
In conclusion, laser ablation coupled in parallel both with elemental and molecular MS is a promising method for the localization and characterization of metal species and their interacting organic molecules in biological samples such as tissue sections. Especially for complex samples and questions in life science research, this novel hyphenation technique opens up new analytical possibilities.
Acknowledgement This work was supported in part by the Cells in Motion Cluster of Excellence (CiM - EXC 1003) Münster, Germany (project FF-2013-17) and by the Interdisciplinary Center for Clinical Research (IZKF) Münster (Grant Cia2/013/13).
DISCUSSION
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The first simultaneous elemental and molecular mass spectrometric imaging approach based on laser ablation sampling has been developed. The presented results demonstrate the benefits of the simultaneous LA-ICP-MS/ APCI-MS setup for spatially resolved speciation analysis. The advantages of LA-ICP-MS including high spatial resolution, low detection limits, species-independent response, large dynamic range and low matrix effects for metal- and heteroelement-containing compounds are combined with information on the chemical identity of the metal species or interacting organic molecules. The simple setup composed of established and mostly commercially available equipment offers flexible ion source and mass analyzer combinations and promises therefore a free choice of the MS instruments depending on target compounds or scientific question.
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Rapid Commun. Mass Spectrom. 2013, 27, 2588–2594
