A research group including Master's Student Takao Yasuda, Associate Professor Yoichi Otsuka, and Professor Michisato Toyoda at the Graduate School of Science, the University of Osaka, enhanced the sensitivity of mass spectrometry imaging technology for investigating chemical component differences among individual cells in biological tissues, developing a new measurement method capable of stable, long-term analysis. The study was published in Analytical Chemistry.
(a) Optical image of the tissue section used in this study.
(b) Image showing the distribution of components obtained through 17 hours of mass spectrometry imaging. The component was putatively assigned as hexosylceramide.
(c) Ion image with a pixel size of 10 µm.
(d) Ion image with a pixel size of 5 µm. Enlarged views of the dashed-line regions in (c) and (d) are shown to the right of each figure, respectively.
Provided by the University of Osaka
Otsuka has previously led the research and development of tapping-mode scanning probe electrospray ionization (t-SPESI) to visualize lipid distributions in biological tissues.
In t-SPESI, a capillary probe (probe), which is a thin glass tube with a tip of several micrometers, is vibrated up and down while a solvent with a high voltage applied flows through its interior. When the tip of the probe contacts the sample surface, a solvent bridge (liquid bridge) forms between the probe and the sample surface, locally extracting sample components. Applying a high voltage to the solvent containing the extracted sample components generates gas-phase ions. The generated ions are introduced into a mass spectrometer and analyzed.
Mass spectrometry imaging can be performed by scanning the probe two-dimensionally across the sample. However, as the spacing between measurement points becomes finer, the number of molecules obtained from a single measurement point decreases, requiring higher detection sensitivity. In addition, measuring a wide area in detail requires a long time, and if the probe tip becomes coated with tissue-derived components during that period, molecular extraction and ionization become unstable. Therefore, achieving both high detection sensitivity and stable molecular extraction and ionization over long periods was a challenge.
Thus, to achieve single-cell mass spectrometry imaging using t-SPESI, the research group miniaturized the measurement apparatus, formed a molecular layer on the capillary probe surface, and evaluated performance using mouse brain tissue sections.
First, they miniaturized the microscope unit and sample stage used to observe the sample position and the position of the liquid bridge formed at the probe tip. By miniaturizing the sample stage, they were able to shorten the metallic ion transfer tube used to introduce ions generated at the probe tip into the mass spectrometer.
Shortening the ion transfer tube suppressed the loss of generated ions before reaching the mass spectrometer, achieving an apparatus configuration capable of detecting even small amounts of molecules obtained from minute measurement points. When evaluating the performance of the apparatus using a sodium iodide solution, they confirmed that the ion signal intensity increased by an average of approximately two times.
Next, to suppress the adhesion of tissue-derived components to the probe surface during long-term measurements, a molecular layer of PFPTES was formed on the surface of the capillary probe. PFPTES is known to have a structure in which fluorine is bonded to a benzene ring and to form covalent bonds with quartz surfaces. By utilizing this property, they believed that a stable molecular layer could be formed on the surface of the quartz capillary probe, imparting water and oil repellency derived from PFPTES molecules to the probe surface.
To confirm that this molecular layer was actually formed on the probe surface, imaging of the probe tip portion was conducted using X-ray photoelectron spectroscopy (XPS). On the probe with the PFPTES molecular layer, an F 1s signal derived from fluorine was clearly observed along the shape of the probe. In addition, imaging by time-of-flight secondary ion mass spectrometry (TOF-SIMS) was also performed.
As a result, on the probe with the PFPTES molecular layer, fragment ions containing fluorine and benzene rings derived from PFPTES were detected. From these results, it was confirmed that the fluorine-containing molecular layer derived from PFPTES was distributed on the capillary probe surface. Furthermore, using the capillary probe with the PFPTES molecular layer, mass spectrometry imaging of mouse brain tissue sections was performed with pixel sizes of 10 µm and 5 µm, obtaining signals derived from multiple lipids.
Focusing on hexosylceramide, a type of lipid, they succeeded in visualizing its localized distribution in the anterior commissure, corpus callosum, and part of the striatum in mouse brain tissue. Particularly, the lipid distribution that appeared as a single region at a pixel size of 10 µm was confirmed to contain finer island-like structures in images obtained at a pixel size of 5 µm. Moreover, no significant fluctuation in ion signals was observed even during measurements lasting approximately 17 hours, and no obvious changes were observed at the probe tip. This demonstrated that surface molecular modification of the probe using PFPTES suppresses the adhesion of tissue-derived components, stabilizing molecular extraction and ionization.
It was shown that single-cell mass spectrometry imaging of biological tissues can be stably performed over long periods. In the future, applying single-cell mass spectrometry imaging via t-SPESI to diseased tissues may clarify at the molecular level how cell-by-cell molecular distributions change with disease.
Otsuka stated, "Single-cell mass spectrometry imaging is a method to visualize in detail the distribution of components contained in cells that make up biological tissues. In this study, we demonstrated that controlling probe surface properties through molecular modification is effective for stabilizing long-term measurements. Moving forward, by utilizing insights from surface science and chemistry to further examine probe shapes, structures, and modifying molecules, we aim to visualize even more detailed molecular distribution information."
Journal Information
Publication: Analytical Chemistry
Title: Development of a Tapping-Mode Scanning Probe Electrospray Ionization Platform for High-Sensitivity and Long-Term Stability in Single-Cell Mass Spectrometry Imaging of Tissue
DOI: 10.1021/acs.analchem.6c02386
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