Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Our protocol enables the visualization of spatial metabolism of microbial interactions that occur during infection. We have developed this imaging mass spectrometry workflow for bacterial co-cultures grown on agar and tissue models. Analyzing non-traditional substrates such as bacterial cultures grown on agar presents many challenges.

Using our protocol, bacterial specimens can be appropriately dried to facilitate MALDI matrix application in subsequent imaging mass spectrometry analysis. This method is broadly applicable to a variety of metabolites, microbial pathogens or diseases, and sample types where a spatial measure of cellular or tissue biochemistry is desired, particularly when studying sample types that require dehydration prior to analysis. After preparing the bacterial culture macrocolonies, remove the agarose bacterial colonies mounted on ITO-coated microscope slides from the packaging material.

Place the samples in a dry box with dessicant for 48 to 72 hours at room temperature. Visually inspect the colonies for deformations in the agar surface. Next, close the vacuum line to the sample chamber, and turn on the power switch to the rotary vane pump to allow the vacuum pump to warm up and achieve proper vacuum pressure.

Turn on the variable voltage transformer to heat the wire filament wrapped around the chamber. Adjust the variable power supply until the internal temperature of the vacuum chamber reaches approximately 50 degrees Celsius. While the pump is warming up, place a slurry of dry ice and 100%ethanol into the cold trap condenser.

Open the vacuum chamber using a wrench to loosen the 16-millimeter double cloth flange clamps. Insert the agarose samples into the chamber, and seal the chamber tightly with the 16-millimeter double cloth flange clamps. Then open the pump valve to evacuate the chamber, and allow the samples to dry for 60 to 120 minutes.

When complete, slowly vent the vacuum chamber to ambient pressure by closing the valve on the rotary vane pump, and opening the vacuum flange to ambient air. Open the chamber as demonstrated previously, and remove the dried agarose sample from the chamber. Use 1, 5-Diaminonaphthalene MALDI matrix for its favorable desorption and ionization of amino acids and negative ion mode.

Attach the sample to the sprayer tray, and load the prepared solutions into the sprayer line. Using the computer software, specify the necessary parameters for a uniform coating of the matrix compound. Monitor the spraying sequence until it is finished.

Remove the sample from the sprayer tray, and store it in a desiccation cabinet until analysis. Insert the matrix-coated sample into the MALDI target plate microscope slide adapter. Inscribe at least three fiducial markers that encompass the sample area using permanent markers.

Use a flatbed scanner to acquire an optical image of the microscope slide including the fiduciary markers. Now define a mass spectrometry instrument method as described in the manuscript. In negative ion mode, select the 100 dalton mass window from mass by charge 100 to 200 for gas phase signal enrichment using a continuous accumulation of selected ions approach which encompasses the mass by charge values of the metabolites of interest.

Open the instrument's image acquisition software, and use the setup wizard to define the file name and location, the MS acquisition mode, regions of interest to be sampled, and the spatial resolution of the image. Teach the sample by using the fiducial markers from the optical image to synchronize with the instrument sample carrier. Then define the regions of interest on the optical image using the software's regions of interest selection tool.

Once all the parameters have been defined, start the acquisition sequence to serially acquire a mass spectrum at each pixel across the defined region of interest. Following image acquisition, save the data with a MIS file extension which is a vendor-specific file format for imaging mass spectrometry platforms. Open the data file in flexImaging, or SCiLS software packages, or export to a non-proprietary data format such as mzML, and visualize using vendor neutral software.

Using the reference software, define mass windows for peaks of interest to generate false color heat maps of ion distributions across the sampled regions. On the average mass spectrum display, zoom in to the peak of interest, and select an appropriate mass window that encompasses the area of the peak profile. Now, right-click on the highlighted mass window, and select Add Mass Filter.

Label the selected mass by charge value using the tentative identification of the suspected metabolite as determined by the high-resolution accurate mass measurement. Select the intensity threshold to adjust the dynamic range of the analyte image displayed by the false color heat map. Further adjust the mass-filtering parameters so that the mass window encompasses the area of the peak profile, and then add the mass filter.

Clean all cryosectioning equipment by rinsing it with 100%ethanol, and allow to dry before placing samples into the cryostat chamber. Prepare ITO-coated microscope slides using an ohmmeter to identify the side of the slide with the conductive coating. Using a diamond-tipped scribe, etch and label the ITO-coated side of the microscope slide.

Then perform cryosectioning on a research cryomicrotome. Transfer the OCT-embedded mouse ceca tissues from a 80 degree Celsius freezer to the cryomicrotome chamber on dry ice. Inside the cryomicrotome chamber, mount the OCT-embedded tissue on a sample chuck using an additional OCT solution.

After the OCT solution has solidified, fix the chuck to the specimen head and begin cryosectioning at 10 to 50 micrometers increments to reach the desired tissue depth or plane of the organ. Once an optimal cross-section of the tissue is reached, begin collecting sections at 12 micrometers thickness. Gently manipulate the slice using artist paintbrushes, and place it onto a Teflon-coated microscope slide.

To pick up the tissue from the Teflon-coated slide, roll the ITO-coated microscope slide on top of the tissue section. Thaw-mount the tissue section to the microscope slide by pressing the palm to the back of the ITO-coated slide. Continue to thaw-mount until the tissue section transitions from translucent to an opaque or matte texture.

For same day analysis, transfer the tissue-mounted microscope slides on dry ice to a dry box with dessicant for storage. Perform the MALDI matrix application and MALDI imaging mass spectrometry as demonstrated earlier. Analyze the ion images from multiple biological replicates, and compare the results for a significance via intensity box plot comparisons using the SCiLS statistical software.

Negative ion mode MALDI analysis of bacterial culture samples allows for detecting dozens of metabolites. High-resolution accurate mass measurements allow for the tentative identification of ornithine and arginine. The measurement regions for imaging mass spectrometry analysis are shown.

Imaging mass spectrometry of these samples allows for the spatial mapping of both ornithine and arginine amino acid metabolites which are found to be altered and differentially localized in the presence of Enterococcus faecalis. The tissue morphology of infected mouse ceca was visualized using hematoxylin and eosin staining. Optical images of mouse ceca tissues after applying a 1, 5-Diaminonaphthalene MALDI matrix layer are shown.

MALDI ion images for ornithine and arginine display unique spatial distributions across the tissue samples. The most important consideration for this protocol is to maintain gentle drying and handling of bacterial culture specimens to minimize bubbling, cracking, and other deformation of the sample. Following imaging mass spectrometry, bright-field and fluorescence microscopy analyses of tissue samples can be performed to evaluate tissue morphology.

This can also enable targeted validation of molecular pathways using antibody-based imaging methods.