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Sample preparation

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Target for MALDI imaging with two conductive-surface microscope slides.

Sample preparation is a critical step in imaging spectroscopy. Scientists take thin tissue slices mounted on conductive microscope slides and apply a suitable MALDI matrix to the tissue, either manually or automatically. Next, the microscope slide is inserted into a MALDI mass spectrometer. The mass spectrometer records the spatial distribution of molecular species such as peptides, proteins or small molecules. Suitable image processing software can be used to import data from the mass spectrometer to allow visualization and comparison with the optical image of the sample. Recent work has also demonstrated the capacity to create three-dimensional molecular images using MALDI imaging technology and comparison of these image volumes to other imaging modalities such as magnetic resonance imaging (MRI).[1][2]

Tissue preparation

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The tissue samples must be preserved quickly in order to reduced molecular degradation. The first step is to freeze the sample by wrapping the sample then submerging it in a cryogenic solution.[3] Once frozen, the samples can be stored below -80°C for up to a year.[3] When ready to be analyzed, the tissue is embedded in a gelatin media which supports the tissue while it is being cut, while reducing contamination that is seen in optimal cutting temperature compound (OCT) techniques.[3] The mounted tissue section thickness varies depending on the tissue.

Tissue sections can then be thaw mounted by placing the sample on the surface of a conductive slide that is of the same temperature, and then slowly warmed from below.[3] The section can also be adhered to the surface of a warm slide by slowly lowering the slide over the cold sample until the sample sticks to the surface.[3]

The sample can then be stained in order to easily target areas of interest, and pretreated with washing in order to remove species that suppress molecules of interest.[3] Washing with varying grades of ethanol removes lipids in tissues that have a high lipid concentration with little delocalization and maintains the integrity of the peptide spatial arrangement within the sample.[3][4]

Matrix application

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The matrix must absorb at the laser wavelength and ionize the analyte. Matrix selection and solvent system relies heavily upon the analyte class desired in imaging. The analyte must be soluble in the solvent in order to mix and recrystallize the matrix. The matrix must have a homogeneous coating in order to increase sensitivity, intensity, and shot-to-shot reproducibility. Minimal solvent is used when applying the matrix in order to avoid delocalization.

One technique is spraying. The matrix is sprayed, as very small droplets, onto the surface of the sample, allowed to dry, and re-coated until there is enough matrix to analyze the sample.[3] The size of the crystals depend on the solvent system used.

Sublimation can also be used to make uniform matrix coatings with very small crystals.[3][5] The matrix is place in a sublimation chamber with the mounted tissue sample inverted above it.[3] Heat is applied to the matrix, causing it to sublime and condense onto the surface of the sample.[3] Controlling the heating time controls the thickness of the matrix on the sample and the size of the crystals formed.[3][5]

Automated spotters are also used by regularly spacing droplets throughout the tissue sample.[3] The image resolution relies on the spacing of the droplets.[3]

Image production

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Images are constructed by plotting ion intensity versus relative position of the data from the sample.[3][6]

Applications

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MALDI-IMS involves the visualization of the spatial distribution of proteins, peptides, lipids, and other small molecules within thin slices of tissue, such as animal or plant.[7][8][9][10] The application of this technique to biological studies has increased significantly since its introduction. MALDI-IMS is providing major contributions to the understanding of diseases, improving diagnostics, and drug delivery. Significant studies are of the eye, cancer research,[11] drug distribution,[12][13] and neuroscience.[14][15]

MALDI-IMS has been able to differentiation between drugs and metabolites and provide histological information in cancer research, which makes it a promising tool for finding new protien biomarkers.[16][17][18] However, this can be challenging because of ion suppression, poor ionization, and low molecular weight matrix fragmentation effects. To combat this, chemical derivatization is used to improve detection.[19][20]

  1. ^ Andersson M, Groseclose MR, Deutch AY, Caprioli RM (2008). "Imaging Mass Spectrometry of Proteins and Peptides: 3D Volume Reconstruction". Nature Methods. 5 (1): 101–108. doi:10.1038/nmeth1145. PMID 18165806.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  2. ^ Sinha TK, Khatib-Shahidi S, Yankeelov TE, Mapara K, Ehtesham M, Cornett DS, Dawant BM, Caprioli RM, Gore JC (2008). "Integrating Spatially Resolved Three-Dimensional MALDI IMS with in vivo Magnetic Resonance Imaging". Nature Methods. 5 (1): 57–59. doi:10.1038/nmeth1147. PMC 2649801. PMID 18084298.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  3. ^ a b c d e f g h i j k l m n o Norris, Jeremy L.; Caprioli, Richard M. (10 April 2013). "Analysis of Tissue Specimens by Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry in Biological and Clinical Research". Chemical Reviews. 113 (4): 2309–2342. doi:10.1021/cr3004295.
  4. ^ Hanreider, J.; Ljungdahl, A.; Faelth, M.; Mammo, S. E.; Bergquist, J.; Andersson, M. (2011). Mol. Cell. Proteomics. 10. {{cite journal}}: Missing or empty |title= (help)
  5. ^ a b Hankin, J. A.; Barkley, R. M.; Murphy, R. C. (2007). J. Am. Soc. Mass Spectrom. 18: 1646. {{cite journal}}: Missing or empty |title= (help)
  6. ^ Spraggins, Jeffrey M.; Caprioli, Richard M. (2011). "High-Speed MALDI-TOF Imaging Mass Spectrometry: Rapid Ion Image Acquisition and Considerations for Next Generation Instrumentation". J. Am. Soc. Mass Spectrom. 22: 1022-1031. doi:10.1007/s13361-011-0121-0.
  7. ^ Caldwell RL, Caprioli RM (2005). "Tissue profiling by mass spectrometry: a review of methodology and applications". Mol. Cell Proteomics. 4 (4): 394–401. doi:10.1074/mcp.R500006-MCP200. PMID 15677390.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  8. ^ Reyzer ML, Caprioli RM (2007). "MALDI-MS-based imaging of small molecules and proteins in tissues". Current Opinion in Chemical Biology. 11 (1): 29–35. doi:10.1016/j.cbpa.2006.11.035. PMID 17185024.
  9. ^ Woods AS, Jackson SN (2006). "Brain tissue lipidomics: direct probing using matrix-assisted laser desorption/ionization mass spectrometry". The AAPS journal. 8 (2): E391–5. doi:10.1208/aapsj080244. PMC 3231574. PMID 16796390.
  10. ^ Khatib-Shahidi S, Andersson M, Herman JL, Gillespie TA, Caprioli RM (2006). "Direct Molecular Analysis of Whole-body Animal Tissue Sections by Imaging MALDI Mass Spectrometry". Analytical Chemistry. 78 (18): 6448–6456. doi:10.1021/ac060788p. PMID 16970320.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  11. ^ Rauser, Sandra; Marquardt, Claudio; Balluff, Benjamin; Deininger, Sören-Oliver; Albers, Christian; Belau, Eckhard; Hartmer, Ralf; Suckau, Detlev; Specht, Katja; Ebert, Matthias Philip; Schmitt, Manfred; Aubele, Michaela; Höfler, Heinz; Walch, Axel (5 April 2010). "Classification of HER2 Receptor Status in Breast Cancer Tissues by MALDI Imaging Mass Spectrometry". Journal of Proteome Research. 9 (4): 1854–1863. doi:10.1021/pr901008d.
  12. ^ Khatib-Shahidi, Sheerin; Andersson, Malin; Herman, Jennifer L.; Gillespie, Todd A.; Caprioli, Richard M. (September 2006). "Direct Molecular Analysis of Whole-Body Animal Tissue Sections by Imaging MALDI Mass Spectrometry". Analytical Chemistry. 78 (18): 6448–6456. doi:10.1021/ac060788p.
  13. ^ Acquadro, Elena; Cabella, Claudia; Ghiani, Simona; Miragoli, Luigi; Bucci, Enrico M.; Corpillo, Davide (April 2009). "Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry Detection of a Magnetic Resonance Imaging Contrast Agent in Mouse Liver". Analytical Chemistry. 81 (7): 2779–2784. doi:10.1021/ac900038y.
  14. ^ Chen, Ruibing; Hui, Limei; Sturm, Robert M.; Li, Lingjun (June 2009). "Three dimensional mapping of neuropeptides and lipids in crustacean brain by mass spectral imaging". Journal of the American Society for Mass Spectrometry. 20 (6): 1068–1077. doi:10.1016/j.jasms.2009.01.017.
  15. ^ Kutz, Kimberly K.; Schmidt, Joshua J.; Li, Lingjun (October 2004). "In Situ Tissue Analysis of Neuropeptides by MALDI FTMS In-Cell Accumulation". Analytical Chemistry. 76 (19): 5630–5640. doi:10.1021/ac049255b.
  16. ^ Stoeckli M, Staab D, Schweitzer A (2006). "Compound and metabolite distribution measured by MALDI mass spectrometric imaging in whole-body tissue sections". International Journal of Mass Spectrometry. 260 (2–3): 195–202. doi:10.1016/j.ijms.2006.10.007.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  17. ^ Castellino, Stephen; Groseclose, M Reid; Wagner, David (November 2011). "MALDI imaging mass spectrometry: bridging biology and chemistry in drug development". Bioanalysis. 3 (21): 2427–2441. doi:10.4155/bio.11.232.
  18. ^ Rauser, Sandra; Marquardt, Claudio; Balluff, Benjamin; Deininger, Sören-Oliver; Albers, Christian; Belau, Eckhard; Hartmer, Ralf; Suckau, Detlev; Specht, Katja; Ebert, Matthias Philip; Schmitt, Manfred; Aubele, Michaela; Höfler, Heinz; Walch, Axel (5 April 2010). "Classification of HER2 Receptor Status in Breast Cancer Tissues by MALDI Imaging Mass Spectrometry". Journal of Proteome Research. 9 (4): 1854–1863. doi:10.1021/pr901008d.
  19. ^ Manier, M. Lisa; Reyzer, Michelle L.; Goh, Anne; Dartois, Veronique; Via, Laura E.; Barry, Clifton E.; Caprioli, Richard M. (13 May 2011). "Reagent Precoated Targets for Rapid In-Tissue Derivatization of the Anti-Tuberculosis Drug Isoniazid Followed by MALDI Imaging Mass Spectrometry". Journal of The American Society for Mass Spectrometry. 22 (8): 1409–1419. doi:10.1007/s13361-011-0150-8.
  20. ^ Franck, J.; El Ayed, M.; Wisztorski, M.; Salzet, M.; Fournier, I. (15 October 2009). "On-Tissue N-Terminal Peptide Derivatizations for Enhancing Protein Identification in MALDI Mass Spectrometric Imaging Strategies". Analytical Chemistry. 81 (20): 8305–8317. doi:10.1021/ac901043n.
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