The Development Of Nanoscale Aperture Tip Desorption Ionization Time-of-Flight Mass Spectrometry And Its Applications In Single-cell Imaging

Cell is the basic structural unit and functional unit in organisms.The study of cellular morphology and composition at high-spatial resolution can provide accurate information about a single cell in a specific microenvironment,which is of great significance for the research of cell signal transduction,disease pathology and biomarkers for early diagnosis of diseases.Mass spectrometry imaging(MSI)has seen a surge in popularity as a biological imaging technique due to its advantages such as label-free and parallel detection of multiple elements/molecules.In particular,high-spatial resolution MSI technology has drawn enormous attention in recent years.Combining the inherent characteristics of MS technology with the elaborate position information provided by high-spatial resolution imaging,high-spatial resolution MSI allows for locating biomolecules at the cell and subcellular level,providing a new visualization tool for in-depth research in the fields of life science and medicine science.As a common direct-sampling technique,laser has long been used as an ion source in mass spectrometry.However,it is hard for laser-based MSI to achieve sub-micron resolution as the low detection sensitivity of mass spectrometry and optical diffraction limits of far-field laser.To this end,we developed a near-field nanoscale aperture tip desorption ionization source(Nano-ATDI)based on atomic force microscope control,and combined it with time-of-flight mass spectrometer,developing a nanoscale aperture tip desorption ionization time-of-flight mass spectrometry(Nano-ATDI-TOFMS).Using this platform,high-spatial resolution imaging of single cell was conducted.The research work of this thesis consists of four parts:introduction to the instrument,preliminary study on imaging performance,single-cell MSI of drugs,and single-cell MSI of nanoparticles.1.Introduction to the instrumentA nanoscale aperture tip desorption ionization time-of-flight mass spectrometer(Nano-ATDI-TOFMS)was developed.The Nano-ATDI-TOFMS consists of a tuning-fork atomic force microscope and a reflective time-of-flight mass spectrometer.This thesis introduces the instrument in detail from the hardware design and software development,including AFM system,nanoscale aperture tip,laser,time-of-flight mass spectrometer of the hardware part,and the imaging software and data processing software of the software part.In order to partially solve the technical obstacles faced by high-resolution MSI,Nano-ATDI-TOFMS used near-field laser to reduce the diameter of the sampling spot,adopted vacuum transmission to improve transmission efficiency,and introduced postionization laser to improve ionization efficiency.This platform can perform three ionization methods:near-field laser desorption ionization(NDI),matrix-assisted near-field laser desorption ionization(MANDI),and near-field laser desorption laser postionization(NDPI),as well as two imaging modes:topographical imaging and mass spectrometry imaging.2.Preliminary study on imaging performance of Nano-ATDI-TOFMSThe sensitivity of NDPI source was firstly discussed.Compared with atmospheric sampling,high-vacuum sampling can effectively reduce the redeposition on sample surface,thereby increasing the transmission efficiency.In addition,the NDI-MS and NDPI-MS analysis of pure sample verified that laser postionization can greatly improve ionization efficiency.The above two points guarantee the sensitivity of the system and lay the foundation for single-cell imaging.Next,spatial resolution of Nano-ATDI-TOFMS was investigated.NDPI-MS and MSI experiments were performed on neutral red coating samples.Lateral resolution of 800 nm and imaging resolution of 860 nm calculated by the crater diameter and 20%-80%rule respectively were obtained.The aperture fiber tip of Nano-ATDI can act as both an ion source and an AFM probe,so the co-registered topographical and chemical imaging of an individual cell can be simultaneously obtained,which shows the multimodal imaging ability at single-cell level.Finally,some typical endogenous compounds of HeLa cells could be detected using this method with MALDI matrix deposited,further widened the analysis range.The Nano-ATDI source developed in this research has high transmission efficiency(vacuum transmission),high ionization efficiency(single photon ionization),and high-spatial resolution(near-field laser).It is also a variant of AFM and has the capability of topographical imaging.All those demonstrate its great analytical potential in single-cell imaging.3.Single-cell imaging of drugs via Nano-ATDI-TOFMSInsights into the pharmacologic effect on cellular processes and the potential toxicological effects are vital to new drug development and evaluation,yet research on these subjects remains a great challenge due to the lack of information regarding the spatiotemporal distribution of drugs and metabolites within a single cell.Herein,NDPI-MS was applied for acquiring the single cell distribution of two structurally similar acridine drugs,proflavine and ethacridine.Subcellular MSI results reveal a concentration-dependent and regional accumulation distribution pattern of acridine drugs.The correlated NDPI-MS and fluorescence imaging further confirms the accumulated location of proflavine and deciphers the ion-trapping accumulation mechanism.Meanwhile,subcellular ethacridine imaging results display a completely different subcellular distribution from that of proflavine,implying structurally similar acridine drugs may possess distinct interaction sites and modes.Furthermore,the possible phenotypic change and fluorescence change of ethacridine after entering cells was demonstrated,which emphasizes the importance of high chemical specificity of high-resolution MSI at the single-cell level.The present results demonstrate the great potential of the integrated single-cell MSI platform for characterizing the drug distribution and its phenotype changes within individual cells,expediting the identification and evaluation of newly developed drugs.4.Single-cell imaging of nanoparticles via Nano-ATDI-TOFMSNanoparticles(NPs)have found prospective applications in the field of biomedicine and biotechnology.Considering that the therapeutic effect of NPs is closely related to their location inside cells,study of the intracellular distribution of NPs in high-spatial resolution is crucial.Herein,NDI-MS was applied to the single-cell imaging of AuNPs and AgNPs at 250 nm/pixel.To determine the location of AuNPs inside cells and further validate this method,the correlated MSI and CLSM imaging were also performed.In addition,simultaneous imaging of nanoparticle and ligands in subcellular lateral resolution was achieved.Nano-ATDI-TOFMS combines the high-spatial resolution of near-field technology with the high specificity and parallel detection ability of mass spectrometry,which provides a powerful approach on insightful exploration of engineered NPs at single-cell level.