Label-free RF Imaging of Cell Membrane Heterogeneity in Liquid

描述（由申请人提供）：细胞膜异质性，如筏（样）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，对异质性的理解却很少。一个主要的困难是缺乏适当的技术活细胞质膜的分子组织检查。目前的技术方法通常需要标记，这会引起伪影，例如光氧化。它们的时间和空间分辨率也往往有限。在该项目中，将开发无标签、非侵入式射频（RF）扫描技术，以填补仪器空白。所获得的RF扫描器具有100 nm或更好的空间分辨率和~ 10 μs的时间分辨率。将获得活的B细胞的筏状脂质结构域中胆固醇、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RF扫描仪的功能是基于在多个频率下测量膜组织的局部电容和电导。据推测，在宽频率范围内的测量捕获分子和结构特定的信息，如弛豫时间和分子组织的分散。然后，逆有效介质理论使目标分子组分和结构的鉴定和分析成为可能。因此，所获得的RF特异性使得能够进行无标记和非侵入性成像。为了实现足够的RF灵敏度，假设 干涉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。为了检验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RF扫描仪。除了液压方法外，还将包括3D电极结构，用于F扫描的电操纵和细胞位置控制。为了确定质膜组织和它们的RF特性之间的定量关系，将表征三元巨单层囊泡（GUV）脂质双层和B细胞质膜的筏（-样）结构域。通过在GUV合成期间调节分子组成和为B细胞补加不同剂量的n-3多不饱和脂肪酸来修饰结构域。将与从常规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RF技术。 拟议研究的主要贡献是一种无标记，非侵入性和超灵敏的RF扫描技术，具有RF特异性，可用于活细胞膜异质性研究。空间分辨率为100 nm或更好，时间分辨率为~10 μs。此外，脂筏（-样）中胆固醇、SPM和n-3 PUFA的浓度 将用RF扫描仪获得活B细胞的区域。

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 μs 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 μs. 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.