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.

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Developing an imaging method with micrometer-to-subcellular resolution is of great significance for visualizing biological samples of different sizes. The label-free and high-throughput mass spectrometry imaging (MSI) technology has shown potential in the implementation of this view. Despite many improvements in MSI witnessed over the past decades, it remains a challenge to achieve a flexible resolution from micrometer down to subcellular level with high detection sensitivity. In this Perspective, we focus on the recent development of MSI techniques based on different ionization resources. Furthermore, several designs of instruments and applications in bioimaging have been reviewed and compared. Additionally, we proposed the perspectives and challenges for MSI methods, including pursuing the matrix free and multiscale resolution with high detection sensitivity and deeply combining machine learning in omics research.

Amyloid fibrillation is known to be associated with degenerative diseases, and mature fibrils are also considered as valuable biomedical materials. Thus, the mechanism and influencing factors of fibrillation have always been the focus of research. However, in vitro studies are always plagued by low reproducibility of kinetics and the molecular mechanism of amyloid fibrillation is under debate until now. Here, we identified the ambient temperature (AT) as a non-negligible interfering factor in in vitro self-assembly of globular protein hen egg-white lysozyme for the first time. By multimodal molecular spectroscopy methods, not only the effect of ATs on the kinetics of protein aggregation was described but also the conformational changes of the molecular structure with different ATs were captured. Through investigating the dependence of interfacial area and catalysis, the reason for this influence was construed by the various aggregation behaviors of protein molecules in the two-phase interface. The results suggest that in vitro mechanism research on protein fibrillation needs to first clarify the AT for a more accurate comparative analysis. The proposal of this concept will provide a new clue for a deeper understanding of the mechanism of protein self-assembly and may have an impact on evaluating the efficiency of amyloid accelerators or inhibitors based on the comparative analysis of protein self-assembly.

Screening appendants on membrane proteins to understand their varied regulation effects is desirable for finding the potential candidates of the membrane-proteintargeted drugs. However, most artificial appendants can hardly support in situ condition screening because they cannot evolve in situ, neither can they send out signals to reflect the modulation. Here, we designed living-DNA appendants to enable such screening. First, the living-cell rolling-circle amplification (LCRCA) strategy was developed to elongate the DNA appendants for self-tangled physical nanogels. The nanogels unify both the functions of membrane-protein modulation and quantification: their sizes increase with the increased time length of LCRCA, which change the regulation effect on the membrane proteins; their large number of repeating short sequences allow quantification of their sizes in the presence of the complementary fluorophore-tagged short DNA. Then, the performance of the living-DNA appendants was examined taking α6β4 integrins as the target, where effective regulation over the distribution of actin filaments, cell viability, and chances of anoikis are all validated. The screening also clearly elucidates the interesting nonlinear relationships between the regulations and the effects. We hope this screening strategy based on living-DNA appendants can stand for a prototype for deeper understanding of natural behaviors of membrane proteins and help in the accurate designing of the membrane-protein-targeted drugs.