Nanometer Resolution Mass Spectro-Microtomography for In-Depth Anatomical Profiling of Single Cells

Abstract

Visually identifying the molecular changes in single cells is of great importance for unraveling fundamental cellular functions as well as disease mechanisms. Herein, we demonstrated a mass spectro-microtomography with an optimal voxel resolution of ∼300 × 300 × 25 nm3, which enables three-dimensional tomography of chemical substances in single cells. This mass imaging method allows for the distinguishment of abundant endogenous and exogenous molecules in subcellular structures. Combined with statistical analysis, we demonstrated this method for spatial metabolomics analysis of drug distribution and subsequent molecular damages caused by intracellular drug action. More interestingly, thanks to the nanoprecision ablation depth (∼12 nm), we realized metabolomics profiling of cell membrane without the interference of cytoplasm and improved the distinction of cancer cells from normal cells. Our current method holds great potential to be a powerful tool for spatially resolved single-cell metabolomics analysis of chemical components during complex biological processes.

Next Generation of Mass Spectrometry Imaging: from Micrometer to Subcellular Resolution

“…Many nuclei were clearly visualized in tissues by endogenous metabolic markers ( m / z 152.1), which provided more opportunities to investigate single-cell spatial metabolomics in different tissues to reveal intercellular interactions and the impact of the tissue microenvironment on cells in situ . At the same time, the subcellular imaging ability of this method was verified by mapping the cytoplasm, nucleus, and nucleolus in a single HeLa cell . Considering the superior depth profiling ability (∼12 nm) of this technology, 3D images of a single cell were also achieved to reveal drug actions more comprehensively.…”

“…Recently, our group developed a tunable matrix-free mass spectrometry imaging approach based on the VUV laser desorption/ionization source (Figure 6b). 34 By optimizing the focusing optical path and laser energies, we can achieve different laser spot sizes from micrometers to nanometers. The ion deflection and focusing electric fields were adjusted to further increase the ion yield and improve the absolute limit of detection sensitivity (ALOD) of endogenous substances up to the ∼amol level.…”

“…Recently, our group developed a tunable matrix-free mass spectrometry imaging approach based on the VUV laser desorption/ionization source (Figure b) . By optimizing the focusing optical path and laser energies, we can achieve different laser spot sizes from micrometers to nanometers.…”

“…One of them is the chemical derivation method for the modification of poorly ionizable molecules. − The number of inorganic nanoparticles and organic molecules have also been discovered to assist in laser desorption ionization. − Integration of postionization technology can further achieve signal amplification of substances. ,, Limited by optical diffraction, the laser wavelengths chosen in traditional LDI MSI methods directly determine a micrometer level of spatial resolution. To further enhance spatial resolution to the subcellular level, the development of near-field laser technology is expected to break through the diffraction limit. − Additionally, the laser wavelength was modulated toward the vacuum extreme or ultraviolet region. − These two ideas have opened other paths to address this problem. Next, we will focus on describing several typical MSI methods based on laser ion sources and their applications in the life sciences.…”

“…Although the EUV source has a high ionization efficiency for organic biomolecules, the high single-photon energy (∼26.4 eV) breaks chemical bonds, which is not conducive to the accurate identification of compounds. Beyond that, a high-intensity VUV laser was also utilized as a desorption/ionization source to ablate the solid biological samples and the imaging capability of both tissue and single cells was verified. − Compared with the SIMS or EUV-MSI technology, this method exhibited fewer ion fragments. Certainly, with the improvement of the imaging resolution, the laser spot size gradually reduces.…”