Label-free RF Imaging of Cell Membrane Heterogeneity in Liquid

描述(由申请人提供)：细胞膜异质性，如RAFT(类)结构域，在各种细胞过程和发病机制中起着重要和积极的作用。然而，人们对这种异质性知之甚少。一个主要的困难是缺乏适当的技术来检测活细胞质膜的分子组织。目前的技术方法通常需要标记，这会导致伪影，如光氧化。它们的时间和空间分辨率也往往是有限的。在这个项目中，将开发无标签、非侵入性射频(RF)扫描技术来填补仪器空白。得到的射频扫描仪具有100 nm或更高的空间分辨率和~10 S的时间分辨率。将获得活的B细胞的RAFT(类)脂类结构域中胆固醇、n-3多不饱和脂肪酸(PUFA)和鞘磷脂(SPM)的相对浓度水平。开发的超灵敏射频扫描仪的功能基于在多个频率下测量膜组织的局部电容和电导。假设在宽频率范围内的测量能够捕捉特定于分子和结构的信息，例如分子组织的驰豫时间和分散程度。然后，反向有效介质理论能够识别和分析目标分子的成分和结构。因此，获得的RF特异性使无标记和非侵入性成像成为可能。为了获得足够的RF灵敏度，假设 干扰过程消除了输出端口处的RF探测波，同时保留了微小的膜域信号。这两个工作假设是基于PI的初步结果和其他研究小组的结果。为了验证由两个工作假设组成的中心假设，将设计、制造和测试超灵敏、高分辨率、多频率的射频扫描仪。除了液压方法外，还将包括一个3D电极结构，用于F扫描的电气操作和细胞位置控制。为了确定质膜结构与其射频特性之间的定量关系，我们将对三元巨单膜泡(GUV)脂双层和B细胞质膜的RAFT(类)结构域进行表征。这些结构域将通过在合成GUV过程中调整分子组成和为B细胞喂食不同剂量的n-3多不饱和脂肪酸来进行修饰。将与传统成像和分子组成分析方法获得的结果进行比较，以帮助验证射频技术。这项研究的主要贡献是一种无标记、非侵入性和具有射频特异性的超灵敏射频扫描技术，用于活细胞膜异质性的研究。空间分辨率为10 0 nm或更高，时间分辨率为~10 S。此外，脂筏中胆固醇、SPM和n-3PUFA的浓度(类) 活的B细胞的结构域将通过射频扫描仪获得。 与公共卫生相关：射频扫描仪为定量描述活细胞-质膜异质性提供了一种关键技术，并有助于了解不同的细胞过程和发病机制。获得的关于B细胞的知识有助于确定n-3多不饱和脂肪酸发挥作用的新机制。

DESCRIPTION (provided by applicant): Cell membrane heterogeneity, such as raft (-like) domains, plays an essential and active role in various cellular processes and pathogeneses. Yet, the heterogeneity is poorly understood. A main difficulty is the lack of appropriate techniques for molecular organization examination of living cell plasma membranes. Current technical approaches often need labeling, which induce artifacts, such as photo-oxidation. Their temporal and spatial resolutions are also often limited. In this project, label-free, non-intrusive radio frequency (RF) scanning techniques will be developed to fill the instrumentation gap. The obtained RF scanners have 100-nm or better spatial resolution and ~ 10 ?s temporal resolution. The relative concentration levels of cholesterol, n-3 polyunsaturated fatty acids (PUFA) and sphingomyelin (SPM) in raft (-like) lipid domains of living B cells will be obtained. The functionality of the developed ultra-sensitive RF scanners is based on measuring local capacitance and conductance of membrane organizations at multiple frequencies. It is hypothesized that measurements over a wide-frequency-range capture molecular- and structure-specific information, such as the relaxation time and dispersion of molecular organizations. Then inverse effective-medium-theory enables the identification and analysis of the targeted molecular components and structures. Thus, the obtained RF specificity enables label-free and non-intrusive imaging. To achieve sufficient RF sensitivity, it is hypothesized that an interference process eliminates RF probing waves at the output-port while preserving minute membrane domain signals. The two working hypotheses are based on the PI's preliminary results and the results from other research groups. To test the central hypothesis, which consists of the two working hypotheses, super- sensitive, high-resolution, multi-frequency RF scanners will be designed, fabricated, and tested. In addition to hydraulic approaches, a 3D electrode structure will be included for electrical manipulation and control of cell positions for F scanning. To identify the quantitative relationship between plasma membrane organizations and their RF properties, the raft (-like) domains of ternary giant-unilamellar-vesicle (GUV) lipid bilayers and B cell plasma membranes will be characterized. The domains will be modified by adjusting molecular compositions during synthesis for GUVs and feeding n-3 polyunsaturated fatty acids of different doses for B cells. Comparisons with the results obtained from conventional imaging and molecular composition analysis methods will be conducted to help verify the RF techniques. The main contribution of the proposed research is a label-free, non-invasive and ultra-sensitive RF scanning technique with RF specificity for living cell membrane heterogeneity studies. The spatial resolution is 100 nm or better, and the temporal resolution is ~10 ?s. Additionally, the concentration of cholesterol, SPM, and n-3 PUFA in the lipid raft (-like) domains of living B cells will be obtained with the RF scanners. PUBLIC HEALTH RELEVANCE: The RF scanners provide a critical technique for quantitative characterization of living-cell-plasma-membrane heterogeneity and facilitate the understanding of diverse cellular processes and pathogeneses. The obtained knowledge on B cells helps identify new mechanisms through which n-3 PUFAs exert their effects.