IDBR: TYPE A: Mass-Sensing Nanostructure-Enhanced Laser Tweezers

非技术描述：了解细胞大小是如何控制的是细胞生物学的一个基本领域，还没有被很好地理解。细胞的大小和质量传达了重要的生理特性，这些特性受到各种环境和遗传因素的密切调控。表征单个细胞的细胞生长率对细胞质量的依赖可以阐明细胞周期进展的潜在机制，也有以细胞体积增大(肥大)为特征的人类疾病的例子，例如可能导致心力衰竭和猝死的心肌肥厚。一般来说，能够随着时间的推移监测单个细胞的大小和质量可以提供重要的生理信息，并在细胞生物学、组织工程、癌症和疾病研究中具有潜在的影响。在哺乳动物细胞中研究大小控制的一个障碍是在测量大小、质量、生长速度以及控制生长和增殖的途径的动力学方面的不准确。细胞质量通常是通过测量细胞大小来间接估计的，然而，已经证明细胞的质量密度在其细胞周期中并不是恒定的。近年来，人们开始研究利用机械谐振器直接测量细胞质量。这些方法要么需要精致的微流控结构和设置，要么仅限于贴壁细胞。机械谐振器上电池的位置也不能精确控制，这限制了传感精度。该项目旨在开发一种精确的细胞质量传感和监测系统，通过将纳米结构增强型激光镊子(NeLT)与一系列MEMS谐振器相结合，可以对贴壁细胞和悬浮细胞进行工作。这个多学科项目将为两名研究生和一名博士后提供培训机会，并将继续实施NSF REU项目，为本科生提供研究经验。国际和平协会和她的团队将继续通过威斯康星大学工程学院探索日为K-12学生参加教育推广活动。这项研究产生的新教材将被纳入外展演示活动中。技术描述：建议的方法利用光子晶体(PHC)平台上的高效光学陷阱，PI的团队已经演示了这一点。该系统将PHC纳米结构集成在MEMS谐振器阵列的表面，实现了活细胞在低光强谐振器上的精确放置。MEMS谐振器由悬挂的微盘结构组成，其谐振频率取决于其质量。细胞质量将通过表征MEMS谐振器的谐振频率来测量。该平台将放置在荧光显微镜下进行光学成像和分析。该系统不需要精密的微流控装置，可同时实现以下功能：(1)适用于贴壁细胞和悬浮细胞。(2)细胞质量随时间的高精度测量，或高通量的及时单点质量测量。(3)作为时间函数的细胞阵列的光学成像，以获得关于细胞状态的尺寸和其他信息。本项目旨在实现以下目标：(A)设计和制备PHC纳米结构，以实现对活细胞的低强度光捕获。利用时域有限差分法(FDTD)模拟来设计和优化PHC纳米结构，以实现对特定尺寸的细胞或颗粒的最大捕获增强。PHC将首先在规则的硅衬底上制造，然后与MEMS谐振器集成。将对不同尺寸的聚苯乙烯微珠进行光学捕获，以确认可以实现更高的捕获效率。(B)设计和制造PHC集成的MEMS谐振器，以实现高精度的质量传感。相同的粒子将被释放并重新捕获在MEMS谐振器上，并重新测量频率响应。将重复这一过程，以评估质量传感的准确性。(C)对贴壁细胞和悬浮细胞进行质量传感和监测。使用血清饥饿、胸腺嘧啶核苷-诺康唑阻断和双胸腺嘧啶核苷阻断来测量细胞质量，以使细胞在细胞周期中同步。为了这项研究，将使用空间光调制器建立可重新配置的光学陷阱阵列。将与该项目一起执行拟议文书的传播计划。该计划包括通过技术会议、出版物和国际和平研究所的研究小组网站传播研究成果和仪器的能力；与密歇根大学商业化中心合作，寻求拟议技术的许可和商业化。

Non-Technical Decscription: Understanding how cell size is controlled is a fundamental area of cell biology that has not been well understood. The size and mass of a cell convey important physiological properties that are closely regulated by various environmental and genetic factors. Characterizing the dependence of cell growth rate on cell mass for individual cells can elucidate the mechanisms underlying cell cycle progression, and there are also examples of human diseases characterized by increased cell size (hypertrophy) such as cardiac hypertrophy that can lead to heart failure and sudden death. In general, being able to monitor size and mass of single cells over time can provide important physiological information and has potential impact in cell biology, tissue engineering, cancer, and disease research. A barrier to studying size control in mammalian cells is the inaccuracy in measuring size, mass, growth rate, and the dynamics of pathways controlling growth and proliferation. Often the cell mass is estimated indirectly by measuring cell size, however, it has been shown that the mass density of a cell is not constant through its cell cycle. Recently, there has been research on using mechanical resonators to measure the cell mass directly. These approaches either require exquisite microfluidic structures and setup or are restricted to adherent cells. The locations of the cells on the mechanical resonators also cannot be accurately controlled, which limits the sensing accuracy. This project aims to develop a precise cell mass sensing and monitoring system that can work with both adherent and suspension cells by combining nanostructure-enhanced laser tweezers (NELT) with an array of MEMS resonators. This multi-disciplinary project will provide training opportunities for two graduate students, one postdoc, and NSF REU program will be pursued to provide research experience for undergraduate students. The PI and her group will continue participating in education outreach activities through UW College of Engineering Discovery Days for K-12 students. New education materials resulting from the research will be incorporated in the outreach demo activities.Technical Decscription: The proposed approach utilizes high efficiency optical trapping on a photonic crystal (PhC) platform that has been demonstrated by the PI's group. The system integrates PhC nanostructures on the surface of an array of MEMS resonators to achieve precise placement of live cells on the resonators with low optical intensity. The MEMS resonator consists of a suspended micro-disk structure whose resonant frequency depends on its mass. Cell mass will be measured by characterizing the resonant frequencies of the MEMS resonators. The platform will be placed under a fluorescence microscope for optical imaging and analysis. This system does not require exquisite microfluidic setup and can simultaneously achieve the following functions: (1) Suitable for both adherent and suspension cells. (2) High-precision measurement of a cell mass versus time, or single-point mass measurement in time with high throughput. (3) Optical imaging of an array of cells as a function of time to obtain size and other information on cell status. This project seeks to achieve the following aims: (A) Design and fabricate PhC nanostructures to achieve optical trapping with low intensity for live cells. Finite-difference time domain (FDTD) simulations will be used to design and optimize the PhC nanostructures to achieve highest trapping enhancement for the specific size of cells or particles. The PhC will be fabricated on a regular Si substrate first then integrated with the MEMS resonators. Optical trapping will be performed for polystyrene beads with various sizes to confirm that enhanced trapping efficiency can be achieved. (B) Design and fabricate PhC-integrated MEMS resonators to achieve mass-sensing with high accuracy. The same particle will be released and re-trapped on the MEMS resonator, and frequency response re-measured. The process will be repeated to allow assessing mass-sensing accuracy. (C) Perform mass sensing and monitoring of adherent and suspension cells. Cells will be synchronized in their cell cycle using serum starvation, thymidine-nocodazole block and double thymidine block for cell mass measurement. An array of reconfigurable optical traps will be set up using a spatial light modulator for the study. A dissemination plan for the proposed instrument will be implemented with the project. The plan involves disseminating the research results and the capability of the instrument through technical conferences, publications, and the PI's research group website; working with UW Center for Commercialization to seek licensing and commercialization of the proposed technology.