MALDI-MSI: What is it, and how does it work?
Matrix-Assisted Laser Desorption/ Ionisation Mass Spectrometry Imaging or, more simply, MALDI-MSI has established itself as one of the most attractive ex vivo (that which is outside of the organism) techniques for the spatial analysis of molecules (analysis which gives information about the molecules’ interactions with their environment) within a given sample . MALDI-MSI is a technique within the wider field of analytical chemistry – the development of tools and methods to measure physical properties of substances and apply those techniques to identify their presence and quantify their amount present. While the term may seem daunting, a breakdown of its components makes the concept seem fairly simple, as Dr. Elizabeth Neumann of UC Davis explains.
Mass spectrometry is an analytical technique in which samples are ionised into their gas-phase ions, are separated in the mass spectrometer according to their mass-to-charge ratio (m/z) and are detected in proportion to their abundance. Based on this information, a mass-to-charge ratio graph is plotted with m/z values on the x-axis and relative intensity on the y-axis (Figure 1); each discrete peak in the graph represents an individual molecule and its abundance in the sample. A mass spectrum of the molecule is thus produced . Mass Spectrometry is used to identify unknown compounds within a sample and to study the structure and chemical properties of different molecules.
Image by Vladislav Andriashvili, “Hexanal molecule”, 2013, via www.commons.wikimedia.org
Another component of MALDI-MSI is the matrix (“matrix-assisted”). It is believed that the function of the matrix essentially is to dilute and isolate analyte (substance under investigation) molecules from each other . This matrix often consists of organic acid, or, more recently, nanoparticles. Spraying is usually the method of application since it forms a homologous layer of matrix atop the sample. Once deposited, matrix particles form co-crystals with the analytes, which lock them in place and make them ready for laser-desorption.
Laser in MALDI-MSI is used to simultaneously vaporise and ionise the analytes . Lasers of both ultraviolet (UV) and infrared (IR) wavelengths are in use, but UV lasers are by far the most important light sources in analytical MALDI . The sample space is divided into grids – each square will be ionised separately by the laser beam and the resulting ions will go through the mass spectrometer.
What can we infer?
Thus, from combining these components, we infer that MALDI-MSI is a soft ionisation technique* that involves a laser striking a matrix of small molecules to convert them into the gas phase without fragmenting or decomposing them, and then making them pass through a mass spectrometer to get a mass spectrum. The resulting mass spectrum can be studied to obtain mass data relating to the analytes and even detect unknown molecules. It is exactly this lucrative technique that Dr. Elizabeth Neumann utilises to study the chemistry behind neurological diseases such as autism, Alzheimer’s, PTSD, and depression.
* Soft ionisation techniques, such as MALDI-MSI, produce little or no fragmentation in ions after ionisation. On the other hand, hard ionisation techniques produce ions with an excess of internal energy, thus causing fragmentation. Here, “fragmentation” refers to the dissociation of unstable molecular ions.
Some applications and limitations
MALDI-MSI has entered the field of tissue-based research by providing unique advantages for analysing tissue specimens in unprecedented detail. A broad spectrum of analytes ranging from proteins, peptides, drugs, pharmaceutical components, lipids, and other analytes are made accessible by this technique . It is the preferable imaging method to study the distribution of drugs and also provides an effortless way to distinguish between active and inactive forms of a given drug as long as they have a different m/z ratio. MALDI-MSI has also been recently used in diagnosis. In the clinic, disease determination is usually done through a limited set of biomarkers, but this approach has difficulties with sensitivity and specificity. Thus, scientists use MSI data instead to create a detection platform for breast cancer in a patient.
While MALDI-MSI allows us to simultaneously determine the distribution of proteins, lipids, metabolites and many other molecules, it suffers from a few limitations as well. The spatial resolution of the image is greatly affected by the size of the laser used; the laser is limited by the diffraction of light and therefore typically does not go beyond 250 nm. The recent use of nanomaterials in matrices is also problematic: they are highly electric and cause shorting by sticking to the electrodes, Dr. Elizabeth Neumann says .
If you’d like to hear more about this exciting analytical technique and one of its avid researchers, visit us on Spotify to listen to our ChemTalk podcast with Dr. Elizabeth Neumann, assistant professor and chemist at the University of California, Davis, to discuss the various types of matrices that may be used in MALDI-MSI, how she first got interested in the technique, and who influenced her on the way.
Find the ChemTalk podcast here: https://open.spotify.com/episode/5KSYKMXQTv2JZfNhYYa7QS
 Science, C. P. (2021). “MALDI Mass Spectrometry Imaging,” in MALDI Mass Spectrometry Imaging and Orbitrap. Editor T. Porta Siegel (Cambridge: Royal Society of Chemistry). doi:10.1039/9781839165191
 “MALDI-TOF Mass Spectrometry”, Creative Proteomics, 17 February 2021.
 Thanh Le. (2018, May 8). MALDI-MSI [Video]. YouTube. https://www.youtube.com/watch?v=8KRLa5GO-II
 Neumann, Elizabeth. Personal Interview. Conducted by Roxanne Salkeld. 13 September 2022.
 Aichler, M., Walch, A. MALDI Imaging mass spectrometry: current frontiers and perspectives in pathology research and practice. Lab Invest 95, 422–431 (2015). https://doi.org/10.1038/labinvest.2014.156