Mass Spec Imaging

Mass spec imaging (MSI) is a technique measuring chemical composition and linking it to spatial coordinates on a surface.  The chemical composition is determined by mass spectrometry, which measures the mass-to-charge ratios (m/z's) of any ions that can be generated from the surface.  Most commonly, the surface is a tissue section on a microscope slide; however, any flat surface could be analyzed given it has suitable dimensions and is properly prepared.  The rest of this document will assume tissue imaging, but much of it could be applied to other surfaces as well.

Every MSI project is unique – there is no standard method that will work for any sample.  The instruments used, sample preparation, data acquisition method, and analysis software will depend on the tissue type and size, the compounds of interest, the spatial resolution needed, and the goals of each experiment.  Additional experiments to assist with or complement MSI may be needed, for example, LC-MS analysis and/or microscopy.

The workflow for MSI depends on the factors above; however, typical preparation of a sample containing pre-sectioned tissue on a slide requires 1-3 hours, followed by 1-10 hours of data acquisition (usually late afternoon/overnight), and then many hours or even a few days of data analysis.  Data acquisition times depend primarily on the area of the analysis region and the spatial resolution required – a larger area or higher spatial resolution will result in longer acquisition times.

Given the above, the MSI facility has adopted a model of user-run samples to ensure experiments are carried out on a project-relevant timeline with the goals of the project in mind during each step.  Researchers who are interested in incorporating MSI into their projects should read this document and follow it up with a phone call or meeting with the facility manager to confirm that the samples are suitable for MSI and that the instrumentation available fits the needs of the project.  Any related MSI literature examples can be brought to the attention of the facility manager at this time, as well.  If the goals of the project and the capabilities of the facility are a fit, then a training session will be scheduled to familiarize new users with the workflows of the various instruments and to discuss how best to proceed with individual projects.  Researchers will then begin bringing samples for their project to prepare and analyze while working closely with the facility staff.  As users become more comfortable with the workflow, they may work more independently.

DESI Source Sprayer

Two instruments in the MSI facility are able to carry out imaging experiments.  These include the Waters Synapt G2-Si QTOF for DESI (desorption electrospray ionization) imaging and the Bruker Ultraflextreme MALDI-TOF/TOF for MALDI (matrix-assisted laser desorption/ionization) imaging.  

Waters Synapt G2-Si QTOF.  The Synapt is a QTOF mass spectrometer that includes IMS (ion mobility spectrometry) for an added dimension of molecular specificity (i.e., collisional cross-section).  Experiments with IMS included are dubbed HDMS in the Waters nomenclature.  Fragmentation by CID (collision-induced dissociation) can be performed either before or after ion mobility separation to gain additional insight or specificity for a single ion (these are HDMS/MS experiments).  The DESI imaging source has a maximum resolution of 50 um, while most experiments are carried out at 70-100 um.  DESI is excellent for observing small molecules and metabolites (typically up to 1000 Da), and requires minimal sample preparation after sectioning onto a clean microscope slide.

Bruker Ultraflextreme MALDI-TOF/TOF.   The Ultraflextreme is a TOF/TOF mass spectrometer that is has both MS and MS/MS capabilities.  Fragmentation on this instrument relies on post-source decay.  The MALDI imaging source has a laser shot frequency of 2 kHz with a maximum resolution of 20 um, and most experiments are carried out at 20-100 um.  MALDI works well for small molecules, larger peptides, and even intact proteins with some optimization.  This instrument requires that tissue sections are on slides with a clear, conductive coating (ITO), and the sample must be evenly sprayed with a MALDI matrix prior to analysis.

HTX TM Sprayer.  This matrix sprayer allows for uniform and consistent coating of samples with a MALDI matrix.  Methods can be optimized with respect to matrix concentration, solvent, solvent and nitrogen flow, and sprayer temperature, height, speed and direction.  The sprayer can also apply a protease (e.g., trypsin) solution for on-tissue digestion.

Leica CM3050 S Cryomicrotome.  This cryomicrotome allows users to bring frozen tissue for OCT-free cryosectioning onto slides for imaging.

Software.  Waters' and Bruker's imaging software, HD Imaging and flexImaging, respectively, are available for image acquisition, viewing, and some analysis.  There is a variety of free and commercial software that has been developed for advanced viewing and analysis of MS images, some of which are currently being tested in the facility.  This will be updated when a software package is chosen for purchase.

For best results, tissue samples should be flash frozen without fixation or embedding.  For particularly fragile tissue (e.g., lung samples), contact facility staff for guidance on mass-spec friendly embedding procedures.  The fixation and embedding process can chemically modify and/or severely contaminate samples such that they are no longer suitable for MSI.  In some cases, for example FFPE samples, the tissue will require extensive washing, which will limit the types of molecules that can be analyzed as many compounds are washed away or delocalized from their original position.  The harvesting and preserving process should be discussed with facility staff before being carried out.

Sectioning.  Just as embedding can severely contaminate samples, so too can the use of OCT (optimal cutting temperature compound) or other adhesives during cryosectioning.  To avoid OCT completely, frozen tissue samples can be attached to the cryostat chuck by pipetting small amounts of water at the point where the tissue and chuck meet.  The water will soon freeze and the tissue will be frozen to the chuck.  If this is not possible, a minimal amount of OCT may be used as a contact between the tissue and the chuck; however, the microtome blade should never touch OCT as it will then smear it across the cutting surface of the tissue.

Sections should be 6-12 um (typically 10 um) thick and should be placed as close to the center of the slide as possible.  If multiple sections will be on one slide, they should be analyzed during the same experiment, and they should be placed side-by-side (not closer than 5 mm together) along the long axis of the slide.

For DESI, a standard clean 25 x 75 x 1 mm slide may be used.  For MALDI, the slide must have a conductive ITO coating on the surface touching the tissue (for example, Delta Technologies part number CG-81IN-S115).

Storage.  If the tissue or tissue sections will be transported or stored for some time, they should be kept on dry ice or in a -80 C freezer.

Leica CM3050S Cryomicrotome