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.
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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.
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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
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DOI: https://doi.org/10.1007/978-1-4939-2987-0_10
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