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High-resolution MALDI mass spectrometric imaging of lipids in the mammalian retina

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Abstract

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) is emerging as a powerful tool for the analysis of molecular distributions in biological samples in situ. When compared to classical histology, the major benefit of this method is the ability to identify and localize many molecules in a single tissue sample. MALDI-MSI spatial resolution currently falls short of traditional microscopic methods as it is limited by instrumentation and sample preparation. Tissue preparation steps, such as matrix deposition, are critical when considering strategies to further enhance the spatial resolution. The mammalian retina was selected as the tissue of choice for method development; its stratified anatomy renders it an ideal tissue to test high-resolution MALDI-MSI as the different layers correspond to specific neuronal classes and cellular structures. We compared alcohol-fixed, paraffin-embedded retina to fresh-frozen samples and matrix that had been deposited by spray or by sublimation. We present a lipid imaging method based on MALDI-MSI of frozen retinal sections with sublimated 2,5-dihydroxybenzoic acid matrix, which results in a highly advanced resolution compared to previous established methods. Hierarchical clustering of the primary data allows robust detection and differentiation of molecular distributions at a spatial resolution between 10 and 20 μm, thus approaching single-cell resolution.

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Abbreviations

DHB:

2,5-Dihydroxybenzoic acid

GCL:

Ganglion cell layer

HPLC:

High-performance liquid chromatography

INL:

Inner nuclear layer

MALDI-MSI:

Matrix-assisted laser desorption/ionization mass spectrometric imaging

MALDI-TOF:

Matrix-assisted laser desorption/ionization time of flight

OCT:

Optimal cutting temperature compound

OPL:

Outer plexiform layer

PALDI:

Nanoparticle-assisted laser desorption/ionization

SEM:

Scanning electron microscope

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Acknowledgments

Nicole Senninger from the Research Unit Protein Science and Claudia-Marieke Pflüger, Ulrike Buchholz, Gabriele Mettenleiter, and Andreas Voss from the Research Unit Analytical Pathology provided technical assistance. This project was funded by Ministry of Education and Research of the Federal Republic of Germany (BMBF) under Project Number 0315508A (SysTec-Verbund IMAGING) to MU and AW, and BMBF Grant Numbers 01IB10004E and 01ZX1310B, and the Deutsche Forschungsgemeinschaft Grant Numbers HO 1258/3-1 and SFB824 TP Z02 to AW.

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Authors and Affiliations

  1. Research Unit Protein Science, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Ingolstädter Landstr. 1, 85764, Neuherberg, Germany

    Alice Ly, Stefanie M. Hauck & Marius Ueffing

  2. Research Unit Analytical Pathology, Institute of Pathology, Helmholtz Zentrum München, German Research Center for Environmental Health (GmbH), Neuherberg, Germany

    Alice Ly, Cédrik Schöne, Stephan Meding, Michaela Aichler & Axel Walch

  3. Bruker Daltonik GmbH, Bremen, Germany

    Michael Becker, Janine Rattke & Detlev Suckau

  4. Adelaide Proteomics Centre, School of Molecular and Biomedical Science, The University of Adelaide, Adelaide, Australia

    Stephan Meding

  5. Center of Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Tübingen, Germany

    Marius Ueffing

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  1. Alice Ly
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  2. Cédrik Schöne
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Correspondence to Marius Ueffing.

Electronic supplementary material

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418_2014_1303_MOESM1_ESM.tif

Supplemenary Figure 1. a. Overall average mass spectra for OCT-embedded fresh-frozen mouse retina washed with 0.1 M phosphate buffer and coated with sublimated DHB matrix. b. Visualizations of individual m/z values from figure 4b. Measured at a 10 µm raster, the different m/z values clearly correspond to distinct retinal layers. Scale bars indicate 100 µm. (TIFF 4339 kb)

418_2014_1303_MOESM2_ESM.tif

Supplemenary Figure 2. a. Overall average mass spectra for fresh-frozen porcine retina coated with sublimated DHB matrix. b. Visualization of m/z 758. 64 from figure 5a. Scale bar indicates 100 µm. (TIFF 3214 kb)

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Ly, A., Schöne, C., Becker, M. et al. High-resolution MALDI mass spectrometric imaging of lipids in the mammalian retina. Histochem Cell Biol 143, 453–462 (2015). https://doi.org/10.1007/s00418-014-1303-1

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