Label-free RF Imaging of Cell Membrane Heterogeneity in Liquid

描述（由申请人提供）：细胞膜异质性，如筏状结构域，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查活细胞膜的分子组织。目前的技术方法通常需要标记，这会引起伪影，如光氧化。它们的时间和空间分辨率也往往有限。在这个项目中，将开发无标签、非侵入式射频（RF）扫描技术来填补仪器空白。所制备的射频扫描器具有100nm以上的空间分辨率和~ 10 μs的时间分辨率。获得活B细胞筏状脂质结构域内胆固醇、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是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可以捕获分子和结构特定的信息，例如分子组织的弛豫时间和弥散。然后利用逆有效介质理论对目标分子组分和结构进行识别和分析。因此，获得的射频特异性使无标签和非侵入性成像成为可能。为了获得足够的射频灵敏度，假设

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.