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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

Abstract

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Introduction

The human microbiome is a highly dynamic ecosystem involving molecular interactions of bacteria, viruses, archaea, and other microbial eukaryotes. While microbial relationships have been intensely studied in recent years, much remains to be understood about microbial processes at the chemical level1,2. This is in part due to the unavailability of tools capable of accurately measuring complex microbial environments. Advances in the field of imaging mass spectrometry (IMS) over the past decade have enabled in situ and label-free spatial mapping of many metabolites, lipids, and proteins in biological sub....

Protocol

NOTE: Animal experiments were approved by the Animal Care and Use Committees of the Children's Hospital of Philadelphia and the University of Pennsylvania Perelman School of Medicine (protocols IAC 18-001316 and 806279).

CAUTION: Clostridium difficile (C. difficile) and Enterococcus faecalis (E. faecalis) are BSLII pathogens and should be handled with extreme caution. Utilize proper decontamination protocols when necessary.

Representative Results

We have performed metabolite MALDI imaging mass spectrometry of model bacterial colonies and mice co-colonized with E. faecalis and C. difficile to study the role of amino acids in microbe-microbe interactions. Bacterial macrocolonies grown on agar serve as a well-defined model to analyze distinct biochemical changes in bacterial biofilm formation. It is important to ensure a controlled drying process for bacterial culture macrocolonies grown on agar media to minimize deformations and cracking in the ag.......

Discussion

During MALDI imaging mass spectrometry, it is important to have a flat sample surface to provide for a consistent focal diameter of the incident MALDI laser on the sample substrate. Deviations in sample height can cause the MALDI laser beam to shift out of focus, causing alterations in beam diameter and intensity, which can affect MALDI ionization efficiency. These alterations in ionization efficiency can result in differences in analyte intensity across the tissue surface that are not reflective of the underlying tissue.......

Acknowledgements

This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) under award GM138660. J.T.S. was supported by the Charles and Monica Burkett Family Summer Fellowship from the University of Florida. J.P.Z. was supported by NIH grants K22AI7220 (NIAID) and R35GM138369 (NIGMS). A.B.S. was supported by the Cell and Molecular Biology Training Grant at the University of Pennsylvania (T32GM07229).

....

Materials

NameCompanyCatalog NumberComments
0.2 μm Titan3 nylon syringe filtersThermo Scientific42225-NN
1,5-diaminonaphthalene MALDI matrixSigma Aldrich2243-62-1
20 mL Henke Ject luer lock syringesHenke Sass Wolf4200.000V0 
275i series convection vacuum gaugeKurt J. Lesker companyKJL275807LL
7T solariX FTICR mass spectrometer equipped with a Smartbeam II Nd:YAG MALDI laser system (2 kHz, 355 nm) Bruker Daltonics
Acetic acid solution, suitable for HPLCSigma Aldrich64-19-7
Acetonitrile, suitable for HPLC, gradient grade, ≥99.9%Sigma Aldrich75-05-8
Ammonium hydroxide solution, 28% NH3 in H2O, ≥99.99% trace metals basisSigma Aldrich1336-21-6
Autoclavable biohazard bags: 55 galGrainger45TV10
Biohazard specimen transport bags (8 x 8 in.)Fisher Scientific01-800-07
Brain heart infusion brothBD Biosciences90003-040
C57BL/6 male mice Jackson Laboratories
CanoScan 9000F Mark II photo and document scannerCanon
CM 3050S research cryomicrotomeLeica Biosystems
Desiccator cabinetSigma AldrichZ268135
Diamond tip scriber, Electron Microscopy Sciences Fisher Scientific50-254-51
Drierite desiccant pelletsDrierite21005
Ethanol, 200 ProofDecon Labs2701
flexImaging softwareBruker Daltonics
ftmsControl softwareBruker Daltonics
Glass vacuum trapSigma AldrichZ549460
HTX M5 TM robotic sprayerHTX Technologies
Indium Tin Oxide (ITO)-coated microscope slidesDelta TechnologiesCG-81IN-S115
In-line HEPA filter to vacuum pumpLABCONCO7386500
Methanol, HPLC GradeFisher Chemical  67-56-1
MTP slide-adapter IIBruker Daltonics235380
Optimal cutting temperature (OCT) compoundFischer Scientific23-730-571
Peridox RTU Sporicide, Disinfectant and CleanerCONTECCR85335 
PTFE (Teflon) printed slides, Electron Microscopy SciencesVWR100488-874
Rotary vane vacuum pump RV8EdwardsA65401903
Tissue-Tek Accu-Edge Disposable High Profile Microtome BladesElectron Microscopy Sciences63068-HP
Transparent vacuum tubingCole PalmerEW-06414-30
Ultragrade 19 vacuum pump oilEdwardsH11025011
Variable voltage transformerPowerstat
Water, suitable for HPLCSigma Aldrich7732-18-5
Wide-mouth dewar flaskSigma AldrichZ120790

References

  1. Biteen, J. S., et al. Tools for the microbiome: nano and beyond. ACS Nano. 10 (1), 6-37 (2016).
  2. Shreiner, A. B., Kao, J. Y., Young, V. B. The gut microbiome in health and in disease. Current Opinion in Gastroenterology. 31

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Microbial CooperationImaging Mass SpectrometryBacterial ColoniesAgarTissue InfectionMALDI MatrixSample PreparationVacuum DryingSpatial MetabolismMetabolitesMicrobial PathogensTissue Biochemistry

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