Single Cell chemical Imaging via nanoscale IR ablation - Mass Spectromety

描述(由申请人提供)：该项目的目标有两个：a)开发一种基于AFM的针尖增强红外消融(NanIR-MS)的纳米级质谱学成像新技术。B)将这一技术应用于单细胞成像。关于细胞内化学成分的信息在理解细胞新陈代谢、分裂、疾病状态、生态效应等方面具有重要意义。由于目前的技术限制，目前对生物系统的大多数分析都是在一组细胞上进行的，假设从一组细胞中得出的总体平均值将产生有用的结果。然而，这通常不是一个有效的假设，因为同一类型的细胞表现出不同的代谢组成，这取决于它们在周期中的阶段、历史和与环境的相互作用。因此，为了检测罕见细胞(例如循环肿瘤)，对细胞进行单独分析是很重要的 细胞)、瞬时细胞状态、细胞环境对细胞和状态的影响以及帮助 在单细胞水平上对基因表达、蛋白质水平和小分子分布的差异的理解。细胞异质性在基因组学、蛋白质组学、脂类组学和代谢组学等在分子水平上表征生物系统的“-omis”领域中尤为显著。这一意义导致美国国立卫生研究院在2011年底推出了一项关于单细胞分析工具的特别关注计划。哺乳动物细胞的大小约为10？m，因此对单个细胞的成像需要至少1？m的成像空间分辨率。纳米IR-MS技术具有至少10倍于此或100 nm的潜在空间分辨率，这为细胞器中的生物分子成像提供了可能性。但要从这一提议中获得创新和商业上的成功，1？m的空间分辨率就足够了。这项提议的具体目的之一是证明纳米IR-MS技术可以应用于单细胞成像。我们将在两种类型的单细胞上证明这一点：来自小鼠大脑的细胞和识别单个循环肿瘤细胞(CTCs)。正如我们的合作者、肿瘤学研究外科医生叶教授在我们的支持信中重申的那样，CTC是主要负责产生转移性疾病的基本实体，目前缺乏鉴定它们的表征技术。为了治愈以上皮为基础的癌症--如乳腺癌、前列腺癌、肺癌、结肠癌和胰腺癌--治疗需要针对那些导致转移的细胞。然而，由于解剖不到或相关的手术并发症，大多数转移性病变从未进行过活检。CTC提供了一种在整个疾病过程中研究转移细胞生物学的容易获得的方法，通常被称为“液体活检”。

DESCRIPTION (provided by applicant): The goals of this project are twofold: a) to develop a new technique for nanoscale Mass spectrometry imaging based on AFM based tip enhanced IR ablation (nanoIR-MS). b) to apply this technique towards the application of Single cell imaging. Information on the chemical composition within a cell has implications in the understanding of cell metabolism, division, disease states, ecological effects etc. Given current technology limitations, most current analyses of biological systems are performed on groups of cells with the assumption that an ensemble average from the group will yield a useful result. However, this typically is not a valid assumption as cells of the same type exhibit diverse metabolic makeup depending on their phase in the cycle, history and interaction with the environment. Thus it is important that cells be analyzed individually in order to detect rare cells (e.g. circulating tumor cells), transient cell states, the influence of the cell environment on cells and states and aid in the understanding of differences in gene expression, protein levels, and small- molecule distributions at the single cell level. Cell heterogeneity is particularly significant in the "-omis" fields such as genomics, proteomics, lipidomics, and metabolomics that characterize biological systems at a molecular level. This significance led to the NIH launching a special focus program on Single cell Analysis Tools in late 2011. The size of mammalian cells is on the order of 10 ¿m and therefore the imaging of single cells requires imaging spatial resolution of at least 1¿m. The nanoIR-MS technology has a potential spatial resolution of at least 10x better than this or 100 nm which offers the possibility of the imaging of biomolecules in organelles. But to achieve an innovative and commercially successful product from this proposal, 1 ¿m spatial resolution would suffice. One of the Specific Aims of this proposal is to demonstrate that the nanoIR-MS technique can be applied for Single Cell Imaging. We will demonstrate this on two types of Single cells: Cells from Mouse brain and also to identify single Circulating Tumor cells (CTCs). As reiterated in our Letter of Support from our collaborator, Prof. Yeh who is an Oncology research surgeon, CTCs are the fundamental entities primarily responsible for spawning metastatic disease and there is a current lack of characterization technologies to identify them . To cure epithelial-based cancers-such as cancers of the breast, prostate, lung, colon and pancreas-therapies need to be directed towards those cells that cause metastases. However, the majority of metastatic lesions are never biopsied due to anatomic inaccessibility or associated morbidity of the procedure. CTCs offer a readily accessible means of studying the biology of metastatic cells throughout the course of disease and are often referred to as 'Liquid Biopsy'.