Mass Spectrometry DOI: 10.1002/anie.200604468 The Chemical Composition of Animal Cells and Their Intracellular Compartments Reconstructed from 3D Mass Spectrometry** Daniel Breitenstein, ChristinaE. Rommel, Rudolf Möllers, Joachim Wegener,* and Birgit Hagenhoff Experimentalapproachesforanalyzingthechemicalcompo- sitionofanimalcellswithspatialresolutionareimportantfor many fields of biomedical research. The analysis of three- dimensional microstructures by time-of-flight secondary-ion massspectrometry(TOF-SIMS)isanemergingtechniqueto make the molecular architecture of biological samples accessible. In SIMS the sample surface is bombarded by primaryions.Afractionoftheenergytransportedintheso- calledcollisioncascadeisdirectedbacktothesamplesurface and causes the desorption of neutral and charged chemical species(secondaryions)fromtheuppermostmolecularlayer. Thesearesubsequentlycollectedandanalyzedwithrespectto theirmass/chargeratio. [1] Today,moststate-of-the-artinstru- ments for organic applications use TOF analyzers for mass determination of the desorbed secondary ions. [2] TOF-SIMS allowsthedetectionofallelementsaswellassmallorganic moleculesinparallelandhasasensitivitydowntotheppm/ femtomole range. [3] Scanning the sample surface with the primary-ion beam provides a 2D image of the chemical surfacecomposition.Moreover,prolongedionbombardment ofthesampleataconstantpositionleadstosputtererosion. Mass analysis of the sputtered material then reveals the verticalcompositionofthesample. [1] Thelateraldistribution oforganicmaterialcanbeimagedwitharesolutionofabout 150–400nm, [4–6] whereas the vertical resolution in organic polymerfilmswasshowntobebetterthan30nm. [7] Application to biological samples like cells and tissues, however, has so far been hindered by the limited signal intensitiesobtainedfromorganicmaterialsandthefactthat the collision cascade destroys organic molecules and, thus, molecular information. The low signal intensities in surface analysis and the loss of molecular information in sputter depthprofilinghavebeenimprovedbytheuseofpolyatomic primaryionslikeAu 3 + andBi 3 + . [3,8] Moreover,buckminster- fullerenes have become available as a new ion source for sputtererosion. [9] TheimpactofC 60 + ionswasfoundtobeless destructivetoorganicsamplesthanthecommonsputterions O 2 + and Cs + . [10] Even intact organic molecules survive the sputterprocess. [11] Thus,itwastheobjectiveofthisstudyto reconstructthemolecularcompositionofanimalcellsinthree dimensions by applying repeated cycles of SIMS analysis of thesamplesurfacefollowedbysputtererosionthatexposesa deeperlayerofthesampletothenextroundofSIMSanalysis (TOF-SIMS 3D microarea analysis). In a dual-beam setup Bi 3 + primary ions were used to determine the chemical composition of the surface, and C 60 + ions were used for intermittentsputtererosion. [12] Six confluent layers of normal rat kidney (NRK) cells, grown on cover slips under ordinary cell-culture conditions, wereanalyzedbyTOF-SIMS3Dmicroareaanalysisafterthe cells had been stabilized by chemical fixation. Chemical fixation is a routine procedure to preserve the structure of biologicalsamplesinhighvacuum. [13] TOF-SIMSresultswere very reproducible and similar for all samples so that data presentationhereisconfinedtoonetypicalexperiment. Figure1illustratesauniqueaspectofTOF-SIMSmicro- area analysis that is very different from optical-image acquisition. At the beginning of the experiment a confluent celllayerispresentontheculturesubstrate.Theuppermost molecularlayer(thicklineinFigure1a)accessibleforSIMS will consist of chemical species originating from the cell surfaces. During intermittent sputter cycles the sample sur- Figure 1. Confluent cells on a growth substrate at the beginning (a) and in the middle (b) of a TOF-SIMS sputter erosion experiment assuming constant sputter rates for all materials. The thick line represents the uppermost molecular layer imaged in SIMS. [*] Dr. C. E. Rommel, PD Dr. J. Wegener Institut für Biochemie Westfälische Wilhelms-Universität Münster Wilhelm-Klemm Strasse 2, 48149 Münster (Germany) Fax: (+ 49)251-833-3206 E-mail: wegenej@uni-muenster.de Dr. D. Breitenstein, Dr. B. Hagenhoff Tascon GmbH Heisenbergstrasse 15, 48149 Münster (Germany) Dr. R. Möllers IonTof GmbH Heisenbergstrasse 15, 48149 Münster (Germany) [**] This study was financially supported by the German Ministry for Education and Research (BMBF) under grant number 0312002A (Nanobiotechnology) and the European Union under grant num- bers FP6-513698 (Toxdrop) and FP6-005045 (Nanobiomaps). The authors acknowledge the help of M. Fartmann for careful revision of the manuscript, S. Grunewald for her expert help with the cell cultures, and both R. Kersting and E. Tallarek for technical advice. We also thank E. Niehuis for helpful discussions and J. Zehnpfen- ning for providing software support. Communications 5332 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Angew. Chem. Int. Ed. 2007, 46, 5332 –5335