TITLE: Spatial metabolomics of in situ, host-microbe interactions (practical guide for combining MALDI-MSI and FISH microscopy on the same section)
AUTHORS: Benedikt Geier, Emilia Sogin , Dolma Michellod, Moritz Janda, Mario Kompauer, Bernhard Spengler, Nicole Dubilier, Manuel Liebeke

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Spatial metabolomics describes the location and chemistry of small molecules involved in metabolic phenotypes, defense molecules and chemical interactions in natural communities. Most current techniques are unable to spatially link the genotype and metabolic phenotype of microorganisms in situ at a scale relevant to microbial interactions. Here, we present a spatial metabolomics pipeline (metaFISH) that combines fluorescence in situ hybridization (FISH) microscopy and high-resolution atmospheric pressure mass spectrometry imaging (AP-MALDI-MSI) to image host-microbe symbioses and their metabolic interactions</span><span style = "font-style:italic;">.</span><span> metaFISH aligns and integrates metabolite and fluorescent images at the micrometer-scale for a spatial assignment of host and symbiont metabolites on the same tissue section. To illustrate the advantages of metaFISH, we mapped the spatial metabolome of a deep-sea mussel and its intracellular symbiotic bacteria at the scale of individual epithelial host cells. Our analytical pipeline revealed metabolic adaptations of the epithelial cells to the intracellular symbionts, a variation in metabolic phenotypes in one symbiont type, and novel symbiosis metabolites. metaFISH provides a culture-independent approach to link metabolic phenotypes to community members in situ – a powerful tool for microbiologists across fields.</span></div></div>

[STEPS]
?. Cryo-fixation and Embeddinga) Dissect tissue samples and submerge in precooled (4°C) 2% w/v carboxymethyl cellulose gel (CMC, Mw ~ 700'000, Sigma-Aldrich Chemie GmbH) and snap-freeze in liquid N2
Make sure the sample is fully covered in CMC gelAvoid air bubbles in CMC gel--> this will cause holes in the frozen blockStore samples at -80°C in sealed bags/containers with a minimum volume of airAvoid air bubbles in CMC gel--> this will cause holes in the frozen blockStore samples at -80°C in sealed bags/containers with a minimum volume of air--> this will cause holes in the frozen block--> storage in unsealed containers causes freeze drying of the CMC over months, leaving a "bubble gum-like" embedding that cannot be sectioned
?. Cryo-sectioninga) Trim CMC around sample (be careful, use single edge 'safety razor blade') with the sample centered in the CMC block, leaving ca. 0.5 cm of CMC around the tissueb) Section tissue at 10-15 µm (we used 10 µm) thickness with a cryostat (Leica CM3050 S, Leica Biosystems Nussloch GmbH) at a chamber temperature of -35 °C and object holder at -22 °C
Leave sample at least over night at ca. -20°C before sectioning --> acclimatization of CMC allows for smoother sectioningAdjust cryostat chamber temperature to ca. 10°C colder than object holder temperature--> acclimatization of CMC allows for smoother sectioning --> this compensates for temperature fluctuations during opening and closing of the cryo-chamber
?. Marking and Microscopya) Warm up slide containers to room temperature b) Mark glass slide very close to tissue with white paint (edding 751®, described previously1) for orientation during image acquisition c) Acquire overview images of the marked tissue in bright-field and autofluorescence of the tissue (if possible)
Using toothpicks or needles to create markings often appear very large under the microscope and cannot be matched as precisely--> attaching a short hair (e.g. eye brow) or animal hair to the tip of a toothpick creates a perfect small brush that also takes up the paint and creates µm-scale markingsIt is essential that the white marker is insoluble by the organic solvents used to spray the ionization matrix to avoid diffusion/ leakage into the tissue--> attaching a short hair (e.g. eye brow) or animal hair to the tip of a toothpick creates a perfect small brush that also takes up the paint and creates µm-scale markings      (e.g. edding 751® works with acetone, methanol, ethanol and acetonitrile)
Printouts of the whole tissue section including the markings are very useful for orientation/ navigation to the often small areas (e.g. specific cells) that will be measured with high-resolution MALDI-MSIFor MALDI-MSI setups where the ROI is determined directly on the tissue, the marker is visible below the ionization matrix and helps to navigate on the tissue to determine the ROIThe marker has a specific molecular signature and autofluorescence, which can help with the virtual alignment of microscopy and MS-imagesFor MALDI-MSI setups where the ROI is determined directly on the tissue, the marker is visible below the ionization matrix and helps to navigate on the tissue to determine the ROIThe marker has a specific molecular signature and autofluorescence, which can help with the virtual alignment of microscopy and MS-images
?. Matrix applicationa)  Dissolve 30 mg ml-1 of 2,5-dihydroxybenzoic acid (DHB; 98% purity, Sigma-Aldrich, Steinheim, Germany) in acetone/water (1:1 v/v) containing 0.1% trifluoroacetic acid (TFA)b) Apply 100 µl of the matrix solution with an ultrafine pneumatic sprayer system with N2 gas (SMALDIPrep, TransMIT GmbH, Giessen, Germany)2  to the tissue sectionc) To locate the field of view and facilitate laser focusing, we drew lines around the matrix-covered tissue section using a red marker
There are many different matrix chemicals, solvents and matrix application systems, ranging from sieves to pneumatic sprayers and sublimation devices. --> Important for high-resolution MALDI-MSI is to find a good trade-off between crystal size (crystals must be smaller than the laser spot) and the extraction/ metabolite signal Questions, which will help choosing a suitable matrix/spaying setup (https://www.ncbi.nlm.nih.gov/pubmed/29682685):--> Important for high-resolution MALDI-MSI is to find a good trade-off between crystal size (crystals must be smaller than the laser spot) and the extraction/ metabolite signal --> Ionization mode: negative or positive? Target molecules: polar or non-polar? Target structures: Do I really need high-spatial resolution? If yes, which spot/crystal size is needed?
?. (AP)-MALDI-MSIa) Atmospheric pressure (AP)-MALDI-MSI measurements were carried out at an experimental ion source setup3, coupled to a Fourier transform orbital trapping mass spectrometer (Q ExactiveTM HF, Thermo Fisher Scientific GmbH, Bremen, Germany)b) The setup contained a 337 nm nitrogen laser, operated at 60 Hz for 500 ms, resulting in 30 shots per pixelc) AP-MALDI-MSI was performed in positive mode for a mass detection range of 400–1200 Da and a mass resolving power of 240,000 (at 200 m/z)d) A specifically developed AP-MALDI-MSI autofocus that was based on a laser triangulation system allowed us to avoid oversampling when imaging at a step size of 3 µm4, 5e) Record measured sample surface with a stereomicroscope (e.g. SMZ25, Nikon, Düsseldorf, Germany) after AP-MALDI-MSI
--> Shooting grids onto the same tissue with different laser energies and using FISH afterwards, helps to determine the suitable trade-off between destructivity of the MALDI-laser, the FISH signals after MALDI-MSI and the necessary spatial resolution
An overview image of the tissue section and the ROI, measured with MALDI-MSI is useful to find the same ROI when imaging the section with the CLSM and when matching fluorescence and MSI imagesmicroscopy images allow to record and measure the distance between ablation spots and to determine the real resolution (also a control for oversampling)
?. Post fixationa) Use tissue tip or cotton swab, wetted with 70% ethanol to carefully wipe away matrix and red marker around the tissueb) Submerge glass slide with matrix-covered tissue section in a 2% PFA/PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4) solution for one hour at room temperature c) Wash off fixative by submerging slide 2x20 min in PBS at room temperatured) Carefully dip slide into 96% Ethanol and air dry afterwards
--> prevents diffusion of red color into the tissue
DHB matrix is water soluble and therefore suited for a simultaneous matrix removal/fixation step. When using matrices, which require stronger organic solvents to dissolve, the tissue can be fixed first in 2% PFA/PBS and the matrix can be removed by washing the slide in 96% ethanol afterwards
?. Thaw mounting of the cryo-sectionsa) Thaw-mount cryo-sections onto coated Polysine® slides (Thermo Scientific)b) Subsequently place slides with sections back into cryostat chamber c) Store slides with tissue sections at -80°C in sealable slide containers with silica granules (e.g. LockMailer™ microscope slide jar, Sigma-Aldrich, Steinheim, Germany)
Using polysine coated slides is essential for cross-linking the tissue/bacterial cells during the PFA post-fixation to the glass slide and ensure proper adhesion during washing and hybridization steps. Depending on the MALDI-MSI setup, conductive ITO-coated slides have to be used, which can also be coated with Polysine manually
Silica granules prevent air moisture condensation on the tissue upon removal from the freezer or fridgeAlternatively, slides can be stored in desiccators at room temperature
?. Fluorescence in situ hybridization (FISH)the protocol was modified after a previous study6a) Encircle dried section with a liquid blocker (PAP-Pen, Science Services) on the glass slide to prevent leakage of the hybridization mixture during incubationb) For the hybridization mixture use 5 ng µl-1 of probe in hybridization buffer (35% formamide (v/v), NaCl 0.9 M, 0.02 M Tris-HCl (pH 7.5), 10% dextran sulfate (w/v), 0.02% (w/v) sodium dodecyl sulfate (SDS), 1% (w/v) Blocking Reagent (Roche, Basel, Switzerland)) c) Use a negative control for unspecific binding, labeled with Cy3, for hybridization on a subsequent tissue section during the same FISH experimentd) Hybridize tissue sections and controls with 20-30 µl of hybridization mixture for 2 h in a saturated formamide-water atmosphere at 46 °C (amounts of hybridization mixture should be sufficient to generously cover tissue section) e) Wash the tissue sections on the slides for 15 min in prewarmed buffer (48°C) (0.1 M NaCl, 0.02 M Tris/HCl pH 8.0, 0.01% SDS, 0.005 M EDTA)f) Dip slides in PBS buffer and 96% ethanol speed up the drying process. Subsequently, the DNA of host and symbiotic bacteria was stained with 4',6-diamidino-2-phenylindole (DAPI) for 3 × 10 min at room temperature. For microscopy, sections were mounted with VECTASHIELD®
Before choosing the fluoresence dyes for the probes, testing the autofluorescence of the tissue --> if possible, avoid chosing probes that lie within the maximum of the autofluorescence of the tissue, this will result in better signal-to-noise values for the probe signalWe used specific 16S rRNA probes to target the symbiotic bacteria containing two fluorophores, at the 5'- and 3'-end of the oligonucleotide for increased sensitivity (biomers.net GmbH, Ulm, Germany)7. --> stronger signals based on the two fluorophoresWe chose probes that are directly connected to fluorophores unlike catalyzed reporter deposition (CARD) FISH probes--> additional steps of the CARD FISH protocol such as permeabilizations, activation, inactivation and a second hybridization can cause stress to the tissue and increase the danger of dislocating/ damaging the post-fixed tissueTo further increase the fluorescence signal or label multiple bacterial phylotypes probes with four fluorescence labels could be used in the same protocol8  --> if possible, avoid chosing probes that lie within the maximum of the autofluorescence of the tissue, this will result in better signal-to-noise values for the probe signalWe used specific 16S rRNA probes to target the symbiotic bacteria containing two fluorophores, at the 5'- and 3'-end of the oligonucleotide for increased sensitivity (biomers.net GmbH, Ulm, Germany)7. --> stronger signals based on the two fluorophoresWe chose probes that are directly connected to fluorophores unlike catalyzed reporter deposition (CARD) FISH probes--> additional steps of the CARD FISH protocol such as permeabilizations, activation, inactivation and a second hybridization can cause stress to the tissue and increase the danger of dislocating/ damaging the post-fixed tissueTo further increase the fluorescence signal or label multiple bacterial phylotypes probes with four fluorescence labels could be used in the same protocol8  --> stronger signals based on the two fluorophoresWe chose probes that are directly connected to fluorophores unlike catalyzed reporter deposition (CARD) FISH probes--> additional steps of the CARD FISH protocol such as permeabilizations, activation, inactivation and a second hybridization can cause stress to the tissue and increase the danger of dislocating/ damaging the post-fixed tissueTo further increase the fluorescence signal or label multiple bacterial phylotypes probes with four fluorescence labels could be used in the same protocol8  --> additional steps of the CARD FISH protocol such as permeabilizations, activation, inactivation and a second hybridization can cause stress to the tissue and increase the danger of dislocating/ damaging the post-fixed tissue
?. Widefield microscopya) If possible, acquire overview tile-scans of the sample in bright field and fluorescent channel after MALDI-MSI, using an (automated tile scanning) epifluorescence microscope
This step is not essential but substantially helps do determine the region, which was measured with MALDI-MSI to guide the CLSM acquisitionIf fluorescence dyes are sensitive to bleaching, only acquire a quick overview image at a low magnification objective lens and short exposure times in the bright field channel--> Comparing the overview bright field microscopy image acquired before MALDI-MSI and FISH with the overview acquired afterwards shows potential changes in the tissue integrity e.g. moved/ washed away tissue pieces
?. Confocal Laser Scanning Microscopy (CLSM)a) Image ROI, imaged with AP-MALDI-MSI with a confocal laser scanning microscope (CLSM) (Zeiss LSM 780) b) When selecting the ROI for CLSM, include tissue areas  around region measured with MALDI-MSI (ca. one tile as a margin around ROI)c) Determine top and bottom plane of the tissue section on at least two diagonal corners of the ROI to compensate for potential tilting and inconsistencies of the slide and tissue section thickness, respectivelyd) Record of multiple z-layers throughout the tissue section
Using the CLSM provides an increased resolution, stronger excitation and the possibility of virtual z-stacking, to determine the fluorescence signals throughout the tissue sectionFor imaging bacteria in tissue sections (tile scanning and z-stacking), we found the magnification of a  20x lens at the CLSM as best trade off, between imaging speed and resolutionParticularly for smaller MALDI-MSI ROIs, higher magnifications could be used For imaging bacteria in tissue sections (tile scanning and z-stacking), we found the magnification of a  20x lens at the CLSM as best trade off, between imaging speed and resolutionParticularly for smaller MALDI-MSI ROIs, higher magnifications could be used --> For dyes that quickly bleach, remember that higher magnifications require more tiles in x y and z, increasing acquisition times substantially
?. [comment]
Although we have not tested different FISH approaches, the possibility of labeling specific genes and transcripts could be particularly useful when working on host-microbe or microbe-microbe interactions. Horizontal gene transfer (HGT) often plays an essential role in such interactions and could change the metabolic capabilities of individual organisms. Consequently, to better interpret metabolite distributions, investigating the genomes through additional omics approaches such as DNA sequencing can show, if the production of specific metabolites is restricted to one partner or even bacterial subpopulations in the symbiosis9. Using this information to design specific probes for individual genes would in combination with MALDI-MSI allow to locate gene specific metabolic phenotypes.