Abstract
For nearly a century, histopathology involved the laborious morphological analyses of tissues stained with broad-spectrum dyes (i.e., eosin to label proteins). With the advent of antibody-labeling, immunostaining (fluorescein and rhodamine for fluorescent labeling) and immunohistochemistry (DAB and hematoxylin), it became possible to identify specific immunological targets in cells and tissue preparations. Technical advances, including the development of monoclonal antibody technology, led to an ever-increasing palate of dyes, both fluorescent and chromatic. This provides an incredibly rich menu of molecular entities that can be visualized and quantified in cells—giving rise to the new discipline of Molecular Pathology. We describe the evolution of two analytical techniques, cytometry and mass spectrometry, which complement histopathological visual analysis by providing automated, cellular-resolution constituent maps. For the first time, laser scanning cytometry (LSC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are combined for the analysis of tissue sections. The utility of the marriage of these techniques is demonstrated by analyzing mouse brains with neuron-specific, genetically encoded, fluorescent proteins. We present a workflow that: (1) can be used with or without expensive matrix deposition methods, (2) uses LSC images to reveal the diverse landscape of neural tissue as well as the matrix, and (3) uses a tissue fixation method compatible with a DNA stain. The proposed workflow can be adapted for a variety of sample preparation and matrix deposition methods.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Traut HF, Papanicolaou GN (1943) Cancer of the uterus: the vaginal smear in its diagnosis. Cal West Med 59:121–122
Kamentsky LA, Burger DE, Gershman RJ, Kamentsky LD, Luther E (1997) Slide-based laser scanning cytometry. Acta Cytol 41:123–143
Shapiro HM (2003) Practical flow cytometry, 4th edn. Wiley-Liss, New York
Bendall SC, Simonds EF, Qiu P et al (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332:687–696
Kamentsky LA, Thorell B (1970) Cell population identification studies. Acta Cytol 14:307–312
Luther E, Kamentsky L, Henriksen M, Holden E (2004) Next-generation laser scanning cytometry. Methods Cell Biol 75:185–218
Pozarowski P, Holden E, Darzynkiewicz Z (2013) Laser scanning cytometry: principles and applications-an update. Methods Mol Biol 931:187–212
Darzynkiewicz Z, Smolewski P, Holden E et al (2011) Laser scanning cytometry for automation of the micronucleus assay. Mutagenesis 26:153–161
Ananthanarayanan V, Deaton RJ, Amatya A et al (2011) Subcellular localization of p27 and prostate cancer recurrence: automated digital microscopy analysis of tissue microarrays. Hum Pathol 42:873–881
Stoeckli M, Farmer TB, Caprioli RM (1999) Automated mass spectrometry imaging with a matrix-assisted laser desorption ionization time-of-flight instrument. J Am Soc Mass Spectrom 10:67–71
Li L, Garden RW, Romanova EV, Sweedler JV (1999) In situ sequencing of peptides from biological tissues and single cells using MALDI-PSD/CID analysis. Anal Chem 71:5451–5458
Li L, Moroz TP, Garden RW, Floyd PD, Weiss KR, Sweedler JV (1998) Mass spectrometric survey of interganglionically transported peptides in Aplysia. Peptides 19:1425–1433
Yew JY, Wang Y, Barteneva N et al (2009) Analysis of neuropeptide expression and localization in adult drosophila melanogaster central nervous system by affinity cell-capture mass spectrometry. J Proteome Res 8:1271–1284
Zavalin A, Yang J, Haase A, Holle A, Caprioli R (2014) Implementation of a Gaussian beam laser and aspheric optics for high spatial resolution MALDI imaging MS. J Am Soc Mass Spectrom 25:1079–1082
Luxembourg SL, Mize TH, McDonnell LA, Heeren RM (2004) High-spatial resolution mass spectrometric imaging of peptide and protein distributions on a surface. Anal Chem 76:5339–5344
Agar NY, Kowalski JM, Kowalski PJ, Wong JH, Agar JN (2010) Tissue preparation for the in situ MALDI MS imaging of proteins, lipids, and small molecules at cellular resolution. Methods Mol Biol 656:415–431
Maier SK, Hahne H, Gholami AM et al (2013) Comprehensive identification of proteins from MALDI imaging. Mol Cell Proteomics 12:2901–2910
Boggio KJ, Obasuyi E, Sugino K, Nelson SB, Agar NY, Agar JN (2011) Recent advances in single-cell MALDI mass spectrometry imaging and potential clinical impact. Expert Rev Proteomics 8:591–604
Seeley EH, Oppenheimer SR, Mi D, Chaurand P, Caprioli RM (2008) Enhancement of protein sensitivity for MALDI imaging mass spectrometry after chemical treatment of tissue sections. J Am Soc Mass Spectrom 19:1069–1077
Dai Y, Whittal RM, Li L (1996) Confocal fluorescence microscopic imaging for investigating the analyte distribution in MALDI matrices. Anal Chem 68:2494–2500
Monroe EB, Jurchen JC, Lee J, Rubakhin SS, Sweedler JV (2005) Vitamin E imaging and localization in the neuronal membrane. J Am Chem Soc 127:12152–12153
Andersson M, Groseclose MR, Deutch AY, Caprioli RM (2008) Imaging mass spectrometry of proteins and peptides: 3D volume reconstruction. Nat Methods 5:101–108
Crecelius AC, Cornett DS, Caprioli RM, Williams B, Dawant BM, Bodenheimer B (2005) Three-dimensional visualization of protein expression in mouse brain structures using imaging mass spectrometry. J Am Soc Mass Spectrom 16:1093–1099
Sinha TK, Khatib-Shahidi S, Yankeelov TE et al (2008) Integrating spatially resolved three-dimensional MALDI IMS with in vivo magnetic resonance imaging. Nat Methods 5:57–59
Bocklitz TW, Creceius AC, Matthaus C et al (2013) Deeper understanding of biological tissue: quantitative correlation of MALDI-TOF and Raman imaging. Anal Chem 85:10829–10834
Feng G, Mellor RH, Bernstein M et al (2000) Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28:41–51
Abdelmoula WM, Carreira RJ, Shyti R et al (2014) Automatic registration of mass spectrometry imaging data sets to the Allen brain atlas. Anal Chem 86:3947–3954
Paxinos G, Franklin KBJ (2004) The mouse brain in stereotaxic coordinates, compact 2nd edition. Amsterdam. Elsevier Academic Press, Boston
Goodwin RJ, Lang AM, Allingham H, Boren M, Pitt AR (2010) Stopping the clock on proteomic degradation by heat treatment at the point of tissue excision. Proteomics 10:1751–1761
Agar NY, Yang HW, Carroll RS, Black PM, Agar JN (2007) Matrix solution fixation: histology-compatible tissue preparation for MALDI mass spectrometry imaging. Anal Chem 79:7416–7423
Chaurand P, Schriver KE, Caprioli RM (2007) Instrument design and characterization for high resolution MALDI-MS imaging of tissue sections. J Mass Spectrom 42:476–489
Trede D, Kobarg JH, Oetjen J, Thiele H, Maass P, Alexandrov T (2012) On the importance of mathematical methods for analysis of MALDI-imaging mass spectrometry data. J Integr Bioinform 9:189
Mascini NE, Heeren RMA (2012) Protein identification in mass-spectrometry imaging. Trac-Trend Anal Chem 40:28–37
McDonnell LA, Walch A, Stoeckli M, Corthals GL (2014) MSiMass list: a public database of identifications for protein MALDI MS imaging. J Proteome Res 13:1138–1142
Agar NYR, Yang HW, Carroll RS, Black PM, Agar JN (2007) Matrix solution fixation: histology-compatible tissue preparation for MALDI mass spectrometry imaging. Anal Chem 79:7416–7423
Gabriel SJ, Schwarzinger C, Schwarzinger B, Panne U, Weidner SM (2014) Matrix segregation as the major cause for sample inhomogeneity in MALDI dried droplet spots. J Am Soc Mass Spectrom 25(8):1356–1363
Koomen JM, Stoeckli M, Caprioli RM (2000) Mapping of surrogate markers of cellular components and structures using laser desorption/ionization mass spectrometry. J Mass Spectrom 35:258–264
Norris JL, Cornett DS, Mobley JA et al (2007) Processing MALDI mass spectra to improve mass spectral direct tissue analysis. Int J Mass Spectrom 260:212–221
Alexandrov T (2012) MALDI imaging mass spectrometry: statistical data analysis and current computational challenges. BMC Bioinformatics 13 Suppl 16:S11
Acknowledgements
This work was made possible by grant 1392 from the Amyotrophic Lateral Sclerosis Society of America to J.A. We would like to acknowledge The Barnett Institute for Biological and Chemical Analysis at Northeastern University as well as the Department of Pharmaceutical Sciences’ Core Imaging Facility. We would like to thank the Brandeis University Animal Care Facility for the maintenance of the transgenic mouse colony, Emily Y. Chen and David DeFilippo for their preliminary work in developing the tissue preparation method.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Rawlins, C.M. et al. (2015). Imaging and Mapping of Tissue Constituents at the Single-Cell Level Using MALDI MSI and Quantitative Laser Scanning Cytometry. In: Singh, A., Chandrasekaran, A. (eds) Single Cell Protein Analysis. Methods in Molecular Biology, vol 1346. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2987-0_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2987-0_10
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2986-3
Online ISBN: 978-1-4939-2987-0
eBook Packages: Springer Protocols