Imaging of Intact Tissue Sections: Moving beyond the Microscope

Abstract

MALDI-imaging MS is a new molecular imaging technologyfor direct in situ analysis of thin tissue sections. Multiple analytes can be monitored simultaneously without prior knowledge of their identities and without the need for target-specific reagents such as antibodies. Imaging MS provides important insights into biological processes because the native distributions of molecules are minimally disturbed, and histological features remain intact throughout the analysis. A wide variety of molecules can be imaged, including proteins, peptides, lipids, drugs, and metabolites. Several specific examples are presented to highlight the utility of the technology. MALDI imaging MS (IMS)4 is an emerging new tool for the analysis of biological and clinical tissue samples. It has been shown to be amenable for the analysis of proteins, peptides (both endogenous and enzymatically produced), lipids, and small molecules (such as drugs and endogenous metabolites). Spatial relationships of molecules within a specimen are preserved because intact tissue is directly analyzed without homogenization. In this way, molecules can be interrogated in their native environments, providing new insights into the biological processes involved.IMS requires minimal sample preparation for analysis. Thin sections of tissue samples (typically, 5-10 m thick) are obtained from frozen or formalin-fixed paraffin-embedded (FFPE) tissue blocks and collected on conductive MALDI targets. A matrix compound (typically, a small organic acid as well as a proteolytic enzyme when necessary) is applied to the surface of the tissue sample. Mass spectra are subsequently collected by firing a laser in an ordered pattern of thousands of ablated spots on the tissue section, and a discrete spectrum is collected from each location on the sample. Each ablated spot is analogous to a pixel in a digital photograph. Each pixel (spectrum) contains many analytes that can be individually displayed as a function of their position and relative intensity within the tissue section. In this way, hundreds of images from specific molecular species can be generated simultaneously from a single tissue section without prior knowledge of their identities. Specific reagents such as antibodies are not needed. Alternatively, IMS can be carried out in a profiling mode in which only relatively small selected areas from each tissue section are targeted for analysis. A stained serial section is typically used to guide the analysis. The sample preparation and analysis process of fresh-frozen and FFPE tissues are summarized in Fig. 1.IMS can be very high-throughput in nature. Often, 10 -20 tissue sections can be collected on a single target plate and analyzed concurrently. With currently available high repetition rate lasers (1 kHz or greater), the entire target plate can be analyzed in a matter of a few minutes to a few hours, depending on the desired spatial resolution. It has recently been shown that, through the use of a 5 kHz laser and continuous laser raster, a rat brain measuring 185 mm 2 can ...

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