The combination of information gained from mass spectrometry (MS) and visualization of spatial distributions in thin sample sections makes this a valuable chemical analysis tool useful for biological characterization. After minimal but careful sample preparation, the general setup of an MSI experiment involves defining an (x, y) grid over the surface of the sample, with the grid area chosen by the user. The mass spectrometer then ionizes the molecules on the surface of the sample and collects a mass spectrum at each pixel on the section, with the resulting spatial resolution defined by the pixel size. After collecting the spectra, computational software can be used to select an individual mass-to-charge (m/z) value, and the intensity of the m/z is extracted from each pixel’s spectrum. These intensities are then combined into a heatmap image depicting the relative distribution of that m/z value throughout the sample’s surface. In order to determine the identity of a specific m/z value, tandem MS (MS/MS) fragmentation can be performed on ions from each pixel, and the fragments can be used to piece together the structure of the unknown molecule. Otherwise, the molecule can be identified based on its intact mass by accurate mass matching to databases of known molecules within a certain mass error range.
With the numerous technological advances in recent years, MSI is becoming a more routine tool in clinical practice and the pharmaceutical industry.4-6 Advances include improvements in reproducible sample preparation to ensure reliable interpretation of data and instrumentation that allows for high acquisition speeds and lower spatial resolution, improving throughput and depth of instrumentation. The credibility of MSI experiments has further been enhanced by the development of methods for absolute quantitation of detected molecules. To help with large computational endeavors, statistical workflows and machine learning algorithms have been implemented to handle the large imaging datasets being produced with modern day instrumentation. MSI can also be combined with other complementary imaging modalities, such as microscopy, Raman spectroscopy, and MRI, to strengthen any biological conclusions. With both hardware and software improvements, 3-dimensional (3D) renderings and even single-cell resolution using MSI are emerging as future frontiers. With all the advances in this field, MSI is rapidly evolving and requires continuous development to match the current demand.
Overall, the aim of this review is to provide an informative resource for those in the MSI community who are interested in improving MSI data quality and analysis or using MSI for novel applications. Particularly, we discuss advances for the last 2 years in sample preparation, instrumentation, quantitation, statistics, and multi-modal imaging that have allowed MSI to emerge as a powerful technique in the clinic. Also, several, novel biological applications are highlighted that demonstrate the potential for the future of the field.
 [P1]No abstract is needed. According to the author guidelines, the first paragraph of the introduction is uploaded when submitted.
 
“Introduction” is not included as a section. Sections are not numbered. A table of contents will be created for us based upon our section titles and break up.