Mass Spectrometry Imaging in the Analysis of Biological Samples

Mass spectrometry imaging is a type of mass spectrometry analysis that maintains and describes the three-dimensional structure of a sample, allowing the spatial distribution of molecules and biomolecules to be established.

Image Credit: Matveev Aleksandr/

Image Credit: Matveev Aleksandr/

For example, the position and concentration of a particular protein within the brain could be established using mass spectrometry imaging, as opposed to performing repeated traditional mass spectrometry operations on each region of the dissected brain when obtaining the same information.

The mechanism of mass spectrometry imaging and some key applications of the method will be discussed in this article.

How Does Mass Spectrometry Imaging Work?

Mass spectrometry is an analytical tool that exploits the unique mass-to-charge ratio of ionized molecules in their identification, as molecules of known mass and charge move towards a detector at a rate that can be calculated. Mass spectrometry imaging utilizes this same principle but is applied to large, whole samples, such as tissues and organs, usually following sectioning into thin slices.

The sectioned sample under investigation is then fixed in place during mass spectrometry imaging, while a laser or other ionizing device is passed over the sample to ionize molecules at highly specific coordinates. The mass-to-charge ratio of the now ionized molecules can then be established using ordinary mass spectrometry techniques, indicating not only the presence and concentration of the molecule but also the specific region from which it originated in the sectioned sample.

This method of ionization is termed matrix-assisted laser desorption/ionization (MALDI) and is the most popularly employed ionization technique in mass spectrometry imaging. MALDI can ionize molecules of a wide range of molecular weights and species, though it requires that the sample be immersed in a matrix of chemicals that assist in ionization.

Another ionization technique termed desorption electrospray ionization (DESI) is also sometimes utilized in mass spectrometry imaging, wherein an electrically charged mist is sprayed at the sample, facilitating ionization of the analyte by charge transfer. Biological samples often require complex preparation to halt enzyme activity and prevent the degradation and delocalization of molecules within the sample.

Preservation methods such as formalin fixation are unsuitable in mass spectrometry imaging as additional cross-linking impedes ionization; thus, samples are flash frozen, and in some cases, adhesive is utilized to assist in mounting.

Applications of Mass Spectrometry Imaging in Biology

Mass spectrometry is useful for analyzing biological samples because it can accurately quantify various molecules, even large biomolecules such as proteins and lipids. Common uses of mass spectrometry in biological research include the identification and quantification of molecules in a mixture, detection of impurities in a sample, analysis of a purified protein, study of the protein content of a cell sample, monitoring of biochemical reactions, sequencing of amino acids, and more.

In pharmaceutical science, the technique has numerous applications span the full spectrum of the drug discovery process. Mass spectrometry is used in target validation to gain information on a pharmaceutical compound's mechanism of action and aid target identification. It is also used to determine the structure of a pharmaceutical compound and gather data on its pharmacokinetic and pharmacodynamic profile.

The advantages associated with recording the spatial distribution of molecules throughout a tissue or other biological sample are obvious, allowing the above applications to an in situ sample. For example, the precise position of a preponderance of drug target receptors could be identified, or the localized accumulation of metabolic products from a particular drug compound.

Comparable alternative techniques for identifying the biodistribution of molecules in a biological system include fluorescence microscopy or autoradiography, in which the target analyte is labeled by a fluorescent or radioactive probe, respectively. The intensity of fluorescence or radioactivity can also indicate target analyte concentration, though both techniques suffer from poorer sensitivity than mass spectrometry imaging, which is also better able to differentiate between similar compounds, such as metabolites of drugs.

Mass spectrometry imaging also avoids significant preparatory steps in which probe-carrying agents are applied to the sample, preferably while living, to observe natural biodistribution. On the other hand, the required fine sectioning of samples precludes examination of living creatures.

The Future of Mass Spectrometry Imaging

Several technical issues unique to mass spectrometry imaging have presented themselves since the development of the technique, such as ion scattering. The process of ionizing a specific region of the biological sample may cause ions to spread and land on the sample elsewhere, providing a false signal. More precise lasers and improvements to the composition of the matrix have somewhat alleviated this problem, though further refinement is an active area of research.

Similarly, most biological samples are adulterated with calibration standards to accurately determine molecule concentration, though this is difficult in the case of sectioned or whole biological samples examined in mass spectrometry imaging. Calibration standards may be incorporated into the matrix where MALDI is used, providing significantly improved sensitivity.

Mass spectrometry imaging can also be combined with other analysis techniques in biology, providing both advantages. For example, labeled antibody probes specific to the target molecule of interest within the sample could be applied before analysis, inserting a known molecule with a good response to ionization into the sample and aiding in target localization.

Similarly, mass spectrometry imaging can be combined with other physical imaging apparatus, such as magnetic resonance imaging (MRI), providing extremely precise structural information that can be combined with the chemical identification abilities of mass spectrometry. Deeper three-dimensional imaging using mass spectrometry imaging is also becoming increasingly realized by greater manipulation of lasers, with a potential future bottleneck being the capture, storage, and analysis of the massive volumes of data gathered by the technique.


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  • Buchberger, A. R., Delaney, K., Johnson, J., & Li, L.. (2018). Mass Spectrometry Imaging: A Review of Emerging Advancements and Future Insights. Analytical Chemistry90(1), 240–265.
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Last Updated: Aug 24, 2023

Michael Greenwood

Written by

Michael Greenwood

Michael graduated from the University of Salford with a Ph.D. in Biochemistry in 2023, and has keen research interests towards nanotechnology and its application to biological systems. Michael has written on a wide range of science communication and news topics within the life sciences and related fields since 2019, and engages extensively with current developments in journal publications.  


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