Matrix assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS) is a fast-developing, high-throughput analytical technique used to visualize the localization of a variety of molecules, including drugs, lipids, peptides and proteins within the biological systems through in situ analysis of tissue sections. A unique feature of imaging mass spectrometry is the ability of combining histopathology with IMS via histology-driven image analysis. Mass spectrometry determines the molecular weight of a compound by measuring the mass to charge ratio (m/z) of its ionized form. Imaging mass spectrometry is able to detect the ion species generated from the surface of solid sample, thus providing information on the identity and localization of the endogenous (lipid, protein, peptide) and exogenous (xenobiotic, metabolite) molecules in situ.
MALDI is a soft ionization technique in which sample molecules (or analyte) are co-crystallized with matrix compound to form sample-matrix crystals. The crystals are hit by a UV laser beam, and the vaporized matrix carries the analyte with it.1 The mass (m) to charge (z) ratio (m/z) of the analyte must then be determined for its identification. To accomplish this, MALDI can be coupled to a time-of-flight (TOF) analyzer in which ions generated by UV vaporization are accelerated by an electrostatic field and subsequently allowed to drift in a neutral field.
Due to acceleration, ions with equal charge (z) have the same kinetic energy. However, ion velocities differ based on their m/z values. Large heavy particles move more slowly through the flight tube and reach the detector in a longer period of time compared with smaller lighter particles. Therefore, the time each ion spends to reach the detector determines its m/z ratio (Wiley and McLaren, 1955). In a typical mass spectrum, the x-axis represents m/z values, whereas the y-axis indicates total ion counts, together reporting relative abundance of individual ions. In IMS, the m/z values of the ions are measured across the tissue to collect molecular information from a regularly spaced array of pixels. The distance between pixels determines spatial resolution of the image. A mass spectrum is generated at each position fired by the laser. The distribution and relative intensity of individual ions can be visualized within the section using a pseudo-color scale (Figure 1). The positions of molecules visualized on the image can be directly correlated to tissue structure and probably function or disease as well.
Proper sample preparation is critical for maintaining the spatial integrity of molecules distributed in tissue, and avoiding delocalization and degradation of analytes. For fresh frozen tissues, typical pretreatment steps include cryostat sectioning at thicknesses between 10 M and 20 M, and thaw-mounting the sections directly onto indium-tin-oxide coated transparent glass slides. Removal of native salts and minerals by washing the sections with an aqueous buffer improves ion intensity signals without loss of spatial resolution (Figure 1). Matrix can be applied to the tissue sections by at least 3 different methods, including with the use of manual systems (airbrush, TLC sprayer), automated devices (matrix spotter, sprayer), or sublimation.
Matrix deposition is an important factor affecting mass resolution, spatial resolution, sensitivity, and reproducibility of MALDI images.2 Manual techniques have been used successfully, but pose challenges due to difficultly controlling matrix application and rendering its thickness and distribution uniform throughout individual samples, and from slide to slide. Automated matrix sprayer devices can reproducibly apply matrix with high resolution and good spectral quality. Sublimation is a dry method that does not require a matrix solvent, and hence avoids analyte delocalization. Furthermore, sublimation produces even layers of small crystals across targets, resulting in very high resolution detection.
IMS data are composed of three components: m/z values of ions, their signal intensities, and x- and y-coordinates. These components are used to visualize localization and intensity of ions throughout the section. Ion intensities within selected regions can be represented as average spectra. Molecular imaging data can be integrated with histology or histopathology for correlative data interpretation by tissue staining with conventional dyes such as Hematoxylin and eosin (H&E) (Figure 1). The best way to accomplish this goal is to post-stain the sections analyzed by IMS, and the immediate adjacent section not used for IMS. The latter helps exclude artefacts caused by IMS acquisition.
Because imaging an entire specimen can generate vast amounts of data that when averaged, can mask critical differences, it is necessary to use data-reduction and refinement approaches. To accomplish this, IMS detailed analyses should be focused on uniformly selected regions of interest (ROI), maintaining the same size and shape of the areas to be analyzed across samples in a given study or experiment. Principal component analysis (PCA) is commonly used to analyze complicated MALDI datasets (Figure 1). PCA reduces the dimensionality of the mass spectra data set to a 2D or 3D coordinate system, where each spectrum is represented by a point. Spectra with similar characteristics are clustered, rendering inter-group differences easily visualized on the PCA plot.3 Validation of IMS results involves preparing and imaging the same samples on separate days using the same conditions of matrix application and IMS acquisition.
Molecular identification of specific ions can be achieved by tandem mass spectrometry analysis (MS/MS) on the tissue sections. To do this, laser energy is used to fragment parent ions (analytes to be identified) and generate product ions collected in MS/MS spectra. Characteristic fragmentation patterns are matched with a database such as LIPID MAPS (Figure 1).
The principal advantage of in situ biochemical imaging by MALDI-IMS poses a major challenge due to the lack of pre-analytical separation of compounds. Consequently, it is difficult to distinguish isobaric and isomeric compounds that have different structures but the same m/z values. However, these problems can be circumvented through the use of high resolution mass analyzers such as orbitrap coupled to MALDI.4 Similarly, ion mobility mass spectrometry can be used to separate and identify isobaric lipids.
In conclusion, MALDI-IMS is a powerful tool for visualizing molecules in tissue sections, and offers the use of “biochemical histopathology” to enhance our understanding of disease. Moreover, this approach enables development of in situ biochemical markers of disease and responses to treatment.
Emine B. Yalcin and Suzanne M. de la Monte are from the liver research center, division of gastroenterology and department of Medicine at Rhode Island Hospital and the Alpert Medical School of Brown University.
1. Di Girolamo, F, Lante, I, Muraca, M and Putignani, L (2013) The Role of Mass Spectrometry in the “Omics” Era. Current organic chemistry 17, 2891-2905.
2. Murphy, RC, Hankin, JA, Barkley, RM and Zemski Berry, KA (2011) MALDI imaging of lipids after matrix sublimation/deposition. Biochimica et biophysica acta 1811, 970-975.
3. Cho, Y-T, Chiang, Y-Y, Shiea, J and Hou, M-F (2012) Combining MALDI-TOF and molecular imaging with principal component analysis for biomarker discovery and clinical diagnosis of cancer. Genomic Medicine, Biomarkers, and Health Sciences 4, 3-6.
4. Landgraf, RR, Prieto Conaway, MC, Garrett, TJ, Stacpoole, PW and Yost, RA (2009) Imaging of lipids in spinal cord using intermediate pressure matrix-assisted laser desorption-linear ion trap/Orbitrap MS. Analytical chemistry 81, 8488-8495.