Anal. Chem. 2004, 76, 4271-4278
Mass Spectrometric Imaging of Lipids in Brain Tissue Peter Sjo 1 vall,*,† Jukka Lausmaa,† and Bjo 1 rn Johansson‡
Department of Chemistry and Materials Technology, SP Swedish National Testing and Research Institute, P.O. Box 857, SE-50115 Borås, Sweden, and Department of Clinical Neuroscience, Karolinska Institutet, SE-17176 Stockholm, Sweden
The spatial distributions of various specific lipids in freeze-dried mouse brain sections were monitored using time-of-flight secondary ion mass spectrometry (TOFSIMS). Mouse brain sections were prepared by cryosectioning, rinsing in 0.15 M NH3HCOO, and freeze-drying, after which the samples were analyzed directly by TOFSIMS, using Au3+ ions as primary ions. Positive and negative TOF-SIMS spectra of the tissue surface contained peaks from quasimolecular ions of a variety of specific lipids, including cholesterol, sulfatides, phosphatidylinositols, and phosphatidylcholines. Images showing the spatial signal intensity distributions of specific ions were recorded across analysis areas ranging from 100 × 100 µm2 to 9 × 9 mm2. The results demonstrate a highly complementary localization of cholesterol and phosphatidylcholine over dimensions from millimeter to micrometer range. Characteristic spatial distributions of several other lipids, including sulfatides and phosphatidylinositols, were observed. Principal component analysis was used to localize regions of the sample surface that show common spectral features. Spectra from different such regions showed large variations in lipid ion signals, indicating large variations in the lipid composition in different regions.
Methods for determining the spatial distribution of specific substances in biological cells and tissue are currently limited by the necessity for chemical labeling (as, for example, in fluorescence microscopy1), insufficient chemical specificity (e.g., FT-IR and Raman microscopy2), and spatial resolution (e.g., imaging MALDI-MS3-5). To gain further knowledge about the molecular structure of cells and tissues, and how structures at different locations are related to biological functions and phenomena, the development and application of new methods for precise chemical analysis at high spatial resolution are therefore highly desirable. * Corresponding author: (e-mail)
[email protected]; (fax) +46 (33) 103388. † SP Swedish National Testing and Research Institute. ‡ Karolinska Institutet. (1) Hell, S. W. Nat. Biotechnol. 2003, 21, 1347-1355. (2) Chenery, D.; Bowring, H. Spectrosc. Eur. 2003, 15, 8-14. (3) Caprioli, R. M.; Farmer, T. B.; Gile, J. Anal. Chem. 1997, 69, 4751-4760. (4) Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M. Nat. Med. 2001, 7, 493-496. (5) Stoeckli, M.; Staab, D.; Staufenbiel, M.; Wiedehold, K.-H.; Signor, L. Anal. Biochem. 2002, 311, 33-39. 10.1021/ac049389p CCC: $27.50 Published on Web 06/26/2004
© 2004 American Chemical Society
In time-of-flight secondary ion mass spectrometry (TOF-SIMS), detailed chemical information of the uppermost molecular layers of a solid surface can be obtained at a submicrometer lateral resolution.6,7 The chemical specificity, lateral resolution (∼200 nm), and surface specificity (∼1-10 nm) make TOF-SIMS an attractive method for the analysis of biological samples.8-11 However, the amount of new biological information obtained by TOF-SIMS has been strongly limited, primarily due to (i) insufficient sensitivity (secondary ion yield), resulting in images with very weak signal intensities, and (ii) extensive fragmentation, preventing the precise identification of large organic molecules. We have recently shown that these limiting factors can be significantly improved upon by using specialized sample preparation procedures, such as chemical imprinting on a silver substrate.12 The development of a new generation of cluster ion sources, notably Au3+ and C60+, has recently been shown to improve the analytical situation significantly when used as primary ion sources in TOF-SIMS; the secondary ion yield of large organic molecules is increased by several orders of magnitude, as compared to when conventional primary ion sources (Ga+, Ar+, etc.) are used. In the case of the Au3+ source, the lateral resolution is only slightly reduced as compared to Ga+ (typically ∼250 nm as compared to ∼150 nm) while the lateral resolution of the C60+ source is ∼3 µm. By using Au3+ as primary ions, a significant improvement in the quality (intensity, contrast, etc.) of TOF-SIMS images of large organic molecules in biological specimens can therefore be expected. The advantages of using a Au cluster primary ion source in the TOF-SIMS analysis of phospholipids and peptides have recently been demonstrated.13 Images showing the lateral distributions of cholesterol and (nonspecific) phospholipids in mouse (6) Pacholski, M. L.; Winograd, N. Chem. Rev. 1999, 99, 2977-3005. (7) Vickerman, J. C., Briggs, D., Eds. ToF-SIMS: Surface Analysis by Mass Spectrometry; IM Publications and SurfaceSpectra Limited: Charlton, Chichester, West Sussex, U.K., 2001. (8) Colliver, T. L.; Brummel, C. L.; Pacholski, M. L.; Swanek, F. D.; Ewing, A. G.; Winograd, N. Anal. Chem. 1997, 69, 2225-2231. (9) McCandlish, C. A.; McMahon, J. M.; Todd, P. J. J. Am. Soc. Mass Spectrom. 2000, 11, 191-199. (10) Pacholski, M. L.; Cannon, D. M., Jr.; Ewing, A. G.; Winograd, N. Rapid Commun. Mass Spectrom. 1998, 12, 1232-1235. (11) Roddy, T. P.; Donald M. Cannon, J.; Ostrowski, S. G.; Winograd, N.; Ewing, A. G. Anal. Chem. 2002, 74, 4020-4026. (12) Sjo¨vall, P.; Lausmaa, J.; Nygren, H.; Carlsson, L.; Malmberg, P. Anal. Chem. 2003, 75, 3429-3434. (13) Touboul, D.; Halgand, F.; Brunelle, A.; Kersting, R.; Tallarek, E.; Hagenhoff, B.; Lapre´vote, O. Anal. Chem. 2004, 76, 1550-1559.
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brain sections at high lateral resolution (
