Photonic Crystal Enhanced Microscopy for Characterization of Cell Attachment

1132301我们提出发展光子晶体增强型显微镜(PCEM)，作为定量和可视化细胞与表面相互作用的一种有效的多模成像方法。PCEM旨在利用光子晶体(PC)生物传感器表面的特性，该表面旨在产生窄带光学共振，用于两个不同的目的。首先，细胞附着改变了PC的共振耦合条件，PCEM能够以足够的分辨率产生细胞附着的空间分布图像，以量化单个细胞内附着强度的变化。其次，PC表面能够有效地耦合到激光照明，从而使PC附近的荧光分子的输出能够增强两个数量级，使用一种名为“PC增强荧光”的技术。PCEM将成为广泛的生命科学研究和药物筛选应用的启动工具，这些应用需要表征细胞外基质材料和细胞膜表达的蛋白质之间的相互作用，以响应细胞生长、促凋亡刺激、强制分化方案、趋化、离子通道激活、跨膜蛋白激活和增殖。PCEM为涉及原代细胞或干细胞的应用提供了有限数量的可用细胞进行分析的能力。拟议的系统建立在第一个PCEM系统的成功实施和演示的基础上，该系统是为涉及DNA微阵列和蛋白质微阵列的应用而设计和建造的。该仪器结合了来自PC上方和下方的物镜耦合照明，用于同时进行无标记成像、亮场成像和增强荧光成像，其光学设计旨在优化PC表面浸泡在细胞介质中时每种模式的灵敏度和分辨率。该系统还结合了二氧化碳环境小室，以便于对细胞进行长时间的持续监测。广泛的影响生物学家正在对细胞膜与表面、化学刺激和其他细胞的相互作用如何调节，以及整合素、离子通道、G偶联跨膜蛋白和丝足虫在迁移、伤口愈合、分化和凋亡等基本过程中的作用有了更深入的了解。然而，目前有几个工具可以对这些过程进行可视化和量化。虽然PCEM最初展示的应用将包括癌细胞细胞毒性、T细胞诱导的凋亡、干细胞分化和心脏细胞刺激，但该系统的能力扩展到任何类型的细胞和任何可以在表面上进行的过程。在伊利诺伊州的环境中，PI是NSF赞助的细胞力学中心(CCMB)IGERT和NIH赞助的癌症纳米技术培训赠款的研究人员，该系统在微纳技术实验室的生物纳米技术实验室内的位置确保了该系统将被纳入多学科培训计划。该系统的用户将由工程学、药理学、动物学、微生物学和免疫学的教职员工、研究生和本科生组成。该项目将开发研究细胞-表面相互作用的通用方法，可广泛用于药物筛选和生命科学研究-扩大光子晶体生物传感器的适用范围。PI在生物传感器仪器的发明、开发和商业化方面有着成功的记录。该提案还描述了一个与该项目的技术目标密切相关的教育计划，该计划将对研究生研究、本科生研究、本科课堂/实验室学习以及向以大学为基础的全女生中学教授生物传感/光子学概念产生影响。PI在让本科生参与他的研究方面有着良好的记录，并在他的ECE416“生物传感器”课程中提供了在NSF资助下开发的技术，无论是以讲座主题的形式还是以基础实验室经验的形式都是如此。

1132301CunninghamIntellectual MeritWe propose the development of a photonic crystal enhanced microscope (PCEM) as apowerful multimode imaging approach for quantification and visualization of the interactionsbetween cells and surfaces. The PCEM is designed to utilize the properties of a photoniccrystal (PC) biosensor surface that is designed to produce narrow bandwidth optical resonancesfor two distinct purposes. First, the resonant coupling condition of the PC is modified by cellattachment, and the PCEM is capable of producing images of the spatial distribution of cellattachment with sufficient resolution to quantify variations in the strength of attachment withinindividual cells. Second, the PC surface enables efficient coupling to laser illumination, resultingin the ability to enhance the output of fluorescent molecules in close proximity to the PC by twoorders of magnitude, using a technique called "PC Enhanced Fluorescence." The PCEM will bean enabling tool for a broad range of life science research and pharmaceutical screeningapplications that require characterization of the interaction between extracellular matrixmaterials and cell membrane-expressed proteins in response to cell growth, pro-apoptoticstimuli, forced differentiation protocols, chemotaxis, ion channel activation, transmembraneprotein activation, and proliferation. PCEM offers the ability to perform assays with a limitednumber of available cells for applications involving primary cells or stem cells. The proposedsystem builds upon the successful implementation and demonstration of the first PCEM system,which was designed and built for applications involving DNA microarrays and proteinmicroarrays. The proposed instrument incorporates objective-coupled illumination from aboveand below the PC for simultaneous label-free imaging, bright field imaging, and enhancedfluorescence imaging with optics designed to optimize the sensitivity and resolution of eachmodality when the PC surface is immersed in cell media. The system also incorporates a CO2environmental chamber to facilitate continuous monitoring of cells for extended periods of time.Broader ImpactsBiologists are developing a more sophisticated understanding of how cell membraneinteractions with surfaces, chemical stimuli, and other cells are modulated, and the role ofintegrins, ion channels, G-coupled transmembrane proteins, and filapodia in fundamentalprocesses such as migration, wound healing, differentiation, and apoptosis. Yet, there are fewtools currently available that allow visualization and quantification of these processes. While theinitial set of demonstrated applications for PCEM will include cancer cell cytotoxicity, T-cellinduced apoptosis, stem cell differentiation, and cardiac cell stimulation, the system'scapabilities extend to any cell type and any process that can be carried out upon a surface. Theenvironment at Illinois, in which the PI is an investigator within the NSF-sponsored Center forCellular Mechanics (CCMB) IGERT, and the NIH-sponsored Cancer Nanotechnology traininggrant, and the location of the system within the Micro and Nanotechnology Laboratory'sBioNanotechnology Laboratory assures that the system will be included in multidisciplinarytraining programs. Users of the system will be comprised of faculty, graduate students, andundergraduate students from Engineering, Pharmacology, Animal Sciences, Microbiology, andImmunology. The project will develop general-purpose methods for studying cell-surfaceinteractions that can be used broadly in pharmaceutical screening and life science research -extending the applicability of photonic crystal biosensors. The PI has a successful track recordof biosensor instrumentation invention, development, and commercialization.The proposal also describes an education plan that is closely linked with the program'stechnical goals with impact upon graduate student research, undergraduate research,undergraduate classroom/laboratory learning, and teaching biosensing/photonics concepts to aUniversity-based all-girls middle school. The PI has a strong track record for involvingundergraduate students in his research, and for making the technology developed under NSFfunding available in his ECE416 "Biosensors" course, both in the form of lecture topics andhands-on laboratory experience.