Differences in Lipid Order and Dynamics in Plasma Membranes Assessed by Nonlinear Optical Microscopy.

When amphiphilic polar dyes were added to the cells, they intercalated predominantly in the outer leaf of the plasma membrane, making them active for second harmonic generation (SHG). The fluorescence of the dye enabled simultaneous 3D imaging of SHG and two-photon excited fluorescence (TPF). Because SHG intensity is sensitive to the alignment of the dyes, which reflects lipid ordering in the plasma membrane, we assessed the difference in lipid ordering by comparing the SHG intensity normalized to the TPF intensity. Together with an enzyme release assay that detects pore formation in the plasma membrane, our SHG assay revealed how polycations affect lipid ordering at low concentrations, where membrane damage has not yet been examined. By scaling the results of the assays with the charge concentration of the two polycations, polyethylenimine (PEI) and poly-l-lysine (PLL), we found that PEI reduced the lipid order more than PLL, and PLL formed more pores than PEI. A comparison of the SHG and TPF images of the wounded cells revealed that one of the lipid dynamics (flip-flop) was significantly enhanced in the bleb membrane. Moreover, the SHG assay indicated that the biocompatible polymer, poly(-(2-hydroxypropyl)methacrylamide), did not affect the lipid order. Thus, our technique allows the assessment of the plasma membrane structure at the molecular level.