Applications of microscope mode imaging mass spectrometry

现代体外诊断缺乏快速识别低浓度蛋白质存在或不存在的精确度。它们灵敏度和吞吐量的提高将使生物分子能够被量化，并导致更有效的蛋白质识别。这项拟议的研究利用一种新的离子显微镜技术--显微质谱学成像(MSI)来捕捉复杂表面的定量和质量分辨的化学快照，从而实现了这些目标。显微镜MSI与传统的微探针MSI的不同之处在于，使用散焦激光或离子束从较大的表面积(~1 cm2)而不是从单个点产生离子。利用特定的电场，产生的离子被它们的m/z分开，并静电聚焦到一个二维探测器阵列上。结果是一系列离子图像，每个m/z一个。这种多路复用方法有可能：a)在一个实验周期内快速识别临床样本中的多个生物标记物；b)通过同时分析大表面来提高数据采集速度；c)通过使用能够记录单个离子事件的成像传感器来提高灵敏度。这项拟议的研究通过三个目标探索这一潜力：第一，通过将反射质谱仪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、质量范围以及空间和质量分辨率；第二，通过演示样品阵列中多个生物标志物的同时分析，最大限度地提高样品吞吐量；第三，通过实施用于标记识别的机器学习，将这些改进应用于复杂生物样品的分析。与NPL合作将使这些目标中的每一个受益。NPL运营着国家质谱学成像卓越中心(NICE-MSI)，并拥有一支由约瑟芬·邦奇教授领导的专门研究团队，他们在质谱仪开发以及MSI在药物发现中的应用方面表现出专业知识。为了更好地了解显微镜MSI的性能和局限性，它将与传统的微探头MALDI方法进行比较，在传统的微探头MALDI方法中，通过在激光下连续移动样品来实现更高的吞吐量，从而缩短没有数据采集的像素之间的时间。这种比较将使我们能够更充分地探索这两种成像模式的基本属性的差异。此外，定量研究将进一步发展微探针MSI在临床相关环境中的应用。了解显微镜模式MSI的检测下限、灵敏度和动态范围将为这项新技术提供坚实的计量学基础。项目目标和里程碑：1)将显微微结构与反射和脉冲提取方法相结合；2)开发用于增强空间分辨率或质量范围的显微离子光学；3)同时进行样品阵列的显微微结构；4)使用质量标签与高通量微探针微结构相比较的组织内蛋白质位置的显微结构。开发合适的机器学习算法5)使用显微镜模式进行定量MSI，并与微探针模式进行比较。这项拟议的研究将受益于牛津大学开发的两项专利技术，这两项技术之前是由EPSRC资助的。第一种是像素成像质谱仪(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周期内对每个解析的m/z进行成像。第二种技术是快速闪烁体，它可以提高典型MSI探测器阵列的时间分辨率。这个项目属于EPSRC物理科学研究领域，属于医学成像、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.