Scanning probe and differential interference contrast imaging methods for studying adhesion-induced tension and membrane fluctuation of red blood cells

 

Natalia Farkas1, Hyeonggon Kang1, Fuyuki Tokumasu2, Jeeseong Hwang3 and

John A. Dagata1

 

1Precision Engineering Division, NIST Gaithersburg , MD 20899

2Laboratory of Malaria and Vector Research, NIAID, NIH, Bethesda, MD 20892-8132

3Optical Technology Division, NIST Gaithersburg , MD 20899

 

 

Red blood cell (RBC) membrane fluctuation mediated by cooperative relationship between its cytoskeleton and lipid bilayer plays an important role in protein dynamics that is indicative of structural-functional properties of healthy or diseased RBCs. Probing of this characteristic membrane behavior requires dynamic interrogation of RBCs under physiological conditions by high-resolution, noninvasive microscopy techniques for which RBCs are required to be immobilized on a substrate while maintaining their viability. Therefore, detailed understanding of the adhesion process and its consequence on RBC shape and dynamic membrane response is critical. In the present study, we demonstrate our ability to engineer substrates with tunable surface zeta potential (SZP) for precise control of RBC adhesion. Specifically, 10 nm gold nanoparticles are adsorbed on poly-L-lysine coated cover slips as a compliant layer to locally modify the non-specific interaction between RBC membrane and substrate. By combining scanning probe microscope (SPM) and differential interference contrast (DIC) imaging techniques we develop a quantitative measurement methodology to investigate the relationship between attachment strength, RBC morphology, cell vibration and membrane fluctuation on these charge and topographically modulated substrates. Adhesion-induced tensing of the RBC membrane on modified substrates leads to changes in cell shape and functionality as determined by SPM force-volume and DIC monitoring of membrane dynamics. The substrate preparation and measurement methods presented here provide a feasible platform to obtain structure-function relationships of viable RBCs under physiological conditions and with that allow us to investigate dynamic behavior of RBCs and their response to diseases.