Applications of microscope mode imaging mass spectrometry

现代体外诊断缺乏精确性，无法快速识别低浓度蛋白质的存在或不存在。其灵敏度和通量的提高将使生物分子能够被定量，并导致更有效的蛋白质识别。拟议的研究解决了这些目标，使用一种新的离子显微镜技术，显微镜质谱成像（MSI），捕捉复杂表面的定量和质量分辨的化学快照。显微镜MSI与传统的微探针MSI不同之处在于使用散焦激光或离子束从大表面积（约1 cm 2）而不是从单个点产生离子。使用特定的电场，产生的离子通过它们的m/z分离，并静电聚焦到二维检测器阵列上。结果是一系列离子图像，每个m/z一个。这种多路复用方法具有以下潜力：a）在单个实验循环期间快速鉴定临床样品中的多种生物标志物; B）通过同时分析大表面来提高数据采集速率，以及c）通过使用能够记录单离子事件的成像传感器来提高灵敏度。拟议的研究通过三个目标探索这一潜力：第一，通过将反射质谱和脉冲离子提取方法结合到离子显微镜中，提高显微镜MSI的灵敏度、质量范围、空间和质量分辨率;第二，通过展示样品阵列中多种生物标志物的同时分析，最大化样品通量;第三个项目将通过实施标记识别的机器学习，将这些改进应用于复杂生物样本的分析。与NPL合作将有利于实现这些目标。NPL运营着国家质谱成像卓越中心（NiCE-MSI），并包含一个由Josephine Bunch教授领导的专门研究团队，他们在质谱仪器开发以及MSI在药物发现中的应用方面具有专业知识。为了更好地了解显微镜MSI的性能和局限性，它将对传统的微探针MALDI方法进行基准测试，在该方法中，通过在激光束下连续移动样品来实现更高的吞吐量，从而减少了没有数据采集的像素之间的时间。这种比较将使这两种成像模式的基本属性的差异进行更充分的探索。此外，定量研究将进一步发展微探针MSI在临床相关背景下的应用。了解显微镜模式MSI的检测限、灵敏度和动态范围将为这项新技术提供坚实的基础。项目目标和里程碑：1）将显微镜MSI与反射器和脉冲提取方法耦合2）开发用于增强空间分辨率或质量范围的显微镜离子光学器件3）样品阵列的同时显微镜MSI 4）与高通量微探针MSI相比，使用质量标签的组织内蛋白质位点的显微镜MSI。5）使用显微镜模式进行定量MSI并与微探针模式进行比较。这项拟议中的研究将受益于牛津大学开发的两项专利技术，该技术此前由EPSRC资助。第一种是像素成像质谱法（PImMS），它使用事件触发的时间戳图像传感器来记录每个检测到的离子的位置和到达时间，精度为12.5 ns，并有效地允许在一个实验MSI周期期间对每个解析的m/z进行成像。第二种技术是快速闪烁体，它提高了典型MSI探测器阵列的时间分辨率。该项目属于EPSRC物理科学研究领域的福尔斯，在医学成像，传感器和仪器，以及分析科学组合，是与NPL的合作。

Modern in vitro diagnostics lack the precision to rapidly identify the presence or absence of proteins at low concentrations. Improvements in their sensitivity and throughput will enable biomolecules to be quantified, and lead to more efficient protein recognition. The proposed research addresses these goals using a novel ion microscopy technique, microscope mass spectrometry imaging (MSI), to capture quantitative and mass-resolved chemical snapshots of complex surfaces. Microscope MSI differs from conventional microprobe MSI in the use of a defocused laser or ion beam to generate ions from large surface areas (~1 cm2) rather than from a single point. Using a specific electric field, the resulting ions are separated by their m/z, and electrostatically focused onto a two-dimensional detector array. The result is a series of ion images, one for each m/z. This multiplexed approach has the potential to: a) rapidly identify multiple biomarkers in clinical samples during a single experimental cycle; b) improve data acquisition rates by simultaneously analysing a large surface, and c) to improve sensitivity by using imaging sensors capable of recording single ion events. The proposed research explores this potential through three objectives: first, improve the sensitivity, mass range, and spatial and mass resolution of microscope MSI by incorporating reflectron mass spectrometry and pulsed ion extraction methods into an ion microscope; second, maximize sample throughput by demonstrating the simultaneous analysis of multiple biomarkers in sample arrays; and the third will apply these improvements to the analysis of complex biological samples by implementing machine learning for marker identification. Partnering with NPL will benefit each of these goals. NPL operates the National Centre of Excellence in Mass Spectrometry Imaging (NiCE-MSI), and contains a dedicated team of researchers, led by Professor Josephine Bunch, who have demonstrated expertise in mass spectrometry instrument development as well as the application of MSI to drug discovery. To better understand the performance and limitations of microscope MSI, it will be benchmarked against conventional microprobe MALDI methods where higher throughput is achieved by continuous movement of a sample under the laser beam, thus reducing the time between pixels where no data are acquired. This comparison will enable a fuller exploration of differences in the fundamental properties of these two imaging modes. Additionally, quantitation studies will further develop the use of microprobe MSI within clinically relevant contexts. Understanding the detection limits, sensitivity and dynamic range of microscope mode MSI will provide a solid metrological foundation for this new technology. Project objectives and Milestones: 1) Coupling microscope MSI with reflectron and pulsed extraction methods 2) Developing microscope ion optics for enhanced spatial resolution or mass range 3) Simultaneous microscope MSI of sample arrays 4) Microscope MSI of protein sites within tissues using mass tags with comparison to high-throughput microprobe MSI. Development of suitable machine learning algorithms 5) Use of microscope mode for quantitative MSI and comparison to microprobe mode. The proposed research will benefit from two patented technologies developed at Oxford and previously funded by the EPSRC. The first, Pixel Imaging Mass Spectrometry (PImMS), uses an event-triggered, time-stamping image sensor to record the position and arrival time for each detected ion to a precision of 12.5 ns, and effectively allows every resolved m/z to be imaged during one experimental MSI cycle. The second technology is a fast scintillator that enhances the time resolution of typical MSI detector arrays. This project falls within the EPSRC Physical Sciences research area, under the medical imaging, sensors and instrumentation, and analytical science portfolios and is a collaboration with NPL.