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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Mentored Quantitative Research Career Development Award (K25)
Project #
5K25GM100480-03
Application #
8733177
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Chin, Jean
Project Start
2012-09-01
Project End
2017-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Clemson University
Department
Engineering (All Types)
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
City
Clemson
State
SC
Country
United States
Zip Code
29634
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