IIBR Instrumentation: Sub-micrometer Resolution Mass Spectrometry Imaging by Deep-UV Laser Ablation and Post-ionization

路易斯安那州立大学和贝勒大学被授予开发一种使用质谱仪对组织中的生物分子进行成像的仪器，该仪器具有高空间分辨率和高质量范围。该仪器简单、低成本、易于使用，将为基础和转译生物学研究中的广泛研究人员带来高性能的非靶向生化成像。该仪器将用于采样和成像相结合的工作流程，能够定位、识别和量化组织中的生物分子。在体外成像方法中，质谱仪具有独特的优势，因为它是非靶向的；因此，它具有无需事先了解成分的情况下检测脂类、代谢物、多肽和蛋白质的能力。该仪器解决了以亚微米精度进行大生物分子电离的关键技术障碍，这是化合物细胞定位所必需的。受益于拟议工具的用户群体包括基础和应用领域的研究人员，这些领域需要对化合物进行空间定位识别和量化。基础生物学研究将受益于一种可以分析各种生化类别的仪器。翻译研究将受益于一种坚固和高度灵敏的仪器，相对较低的成本和易用性将把它带到通常不具备这种能力的研究中心。质谱学成像项目将利用STEM教育资源，用于高中、本科生、研究生和博士后教育和培训。该项目将为参加国家科学基金会S-STEM学者计划和路易斯安那州路易斯·斯托克斯少数民族参与联盟的学生提供研究机会。参加路易斯安那州生物医学研究网络暑期研究项目的本科生研究人员和当地初中和高中的科学教师将通过路易斯安那州立大学凯恩STEM文学中心获得机会。目前的质谱学成像技术需要在二次离子质谱仪(SIMS)和基质辅助激光解吸电离(MALDI)之间进行折衷，二次离子质谱仪(SIMS)使用精确聚焦的离子束来成像小分子，基质辅助激光解吸电离(MALDI)使用聚焦较少但更温和的激光束来成像大分子。这项拟议的研究通过使用高度聚焦的深紫外光消融激光和用于双激光MALDI(MALDI-2)的紫外后电离激光相结合，消除了对空间分辨率和质量范围的妥协。该方法利用消融组织和基质材料上的MALDI效应，因此具有MALDI的质量范围和温和的电离特性。此外，它使用简单、强大和相对低成本的质谱学技术，有可能将该仪器的能力带给更多的生物成像研究人员。MALDI-2成像与深紫外光激光烧蚀进样相结合，用于液相色谱串联质谱分析。将利用各种社交媒体平台以及出版物和会议介绍的传统方法传播项目成果。将通过一个专门的网站和社交媒体在网上发布关于该仪器的照片、视频和最新情况。该网站将与移动设备兼容，展示运行中的仪器的照片和视频以及获得的数据。该网站还将包括指令和协议、示例数据以及仪器控制和分析软件。该项目的预期成果是一个空间分辨率为1微米、质量范围大于10,000 Da的深紫外光消融MALDI-2成像质谱仪的原型。该仪器将能够以与传统MALDI成像质谱计相媲美的速度、样品制备和数据采集的简便性进行成像。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

An award is made to Louisiana State University and Baylor University to develop an instrument for imaging biomolecules in tissue that uses mass spectrometry and has both high spatial resolution and high mass range. The instrument is simple, low-cost, and easy to use and will bring high-performance untargeted biochemical imaging to a broad range of researchers in fundamental and translational biological research. This instrument will be used in a combined sampling and imaging workflow with the ability to localize, identify, and quantify biomolecules in tissue. Mass spectrometry has the unique advantage among in vitro imaging methods in that it is un-targeted; thus, it has the capability of detecting lipids, metabolites, peptides and proteins without prior information about the components. This instrument addresses a critical technological barrier of large biomolecule ionization at submicrometer precision that is necessary for cellular localization of compounds. The user community benefiting from the proposed instrument includes researchers in fundamental and applied areas that require spatially localized chemical compound identification and quantification. Basic biological research will benefit from an instrument that can analyze a wide range of biochemical classes. Translational research will benefit from an instrument that is robust and highly sensitive and the relatively low cost and ease of use will bring it to research centers where this capability is not typically found. The mass spectrometry imaging project will leverage STEM education resources for high-school, undergraduate, graduate, and postdoctoral education and training. The project will provide research opportunities for students participating in the NSF S-STEM Scholars program and the Louis Stokes Louisiana Alliance for Minority Participation. Opportunities will be available for undergraduate researchers from the State participating in the Louisiana Biomedical Research Network summer research program and for local middle and high school science teachers through the LSU Cain Center for STEM Literacy.Current mass spectrometry imaging techniques require a compromise between secondary ion mass spectrometry (SIMS) that uses a precisely focused beam of ions to image small molecules and matrix-assisted laser desorption ionization (MALDI) that uses a less-focused but gentler laser beam to image large molecules. The proposed research eliminates the need for this spatial resolution and mass range compromise through the use of a highly focused deep-UV ablation laser combined with an ultraviolet post-ionization laser for two-laser MALDI (MALDI-2). The approach uses the MALDI effect on ablated tissue and matrix material and therefore has the mass range and gentle ionization properties of MALDI. Furthermore, it uses simple, robust, and relatively low-cost mass spectrometry technology that has the potential to bring the capabilities of the instrument to more biological imaging researchers. MALDI-2 imaging is combined with deep-UV laser ablation sampling for liquid chromatography tandem mass spectrometry analysis. The results of the project will be disseminated using a variety of social media platforms as well as traditional methods of publications and conference presentations An online presence will be established through a dedicated website and social media that will post photos, videos, and updates on the instrument. The website will be mobile device compatible and feature photos and videos of the instrument in operation as well as the data obtained. The site will also include instructions and protocols, example data, and instrument control and analysis software. The anticipated outcome of this project is a prototype deep-UV ablation MALDI-2 imaging mass spectrometer with 1 µm spatial resolution and greater than 10,000 Da mass range. The instrument will be capable of imaging with the speed, ease of sample preparation, and ease of data acquisition that is comparable to conventional MALDI imaging mass spectrometry.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.