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

Area of Science:

• Cell Biology
• Biophysics
• Materials Science

Background:

• The plasma membrane's structure and lipid order are crucial for cellular functions.
• Assessing membrane integrity and molecular dynamics at the nanoscale is challenging.
• Polycations are known to interact with cell membranes, but their effects at low concentrations are not fully understood.

Purpose of the Study:

• To develop and validate a novel imaging technique for assessing plasma membrane lipid ordering and structure.
• To investigate the effects of polycations (PEI and PLL) on membrane lipid order and pore formation at low concentrations.
• To evaluate the impact of a biocompatible polymer on membrane structure.

Main Methods:

• Utilizing amphiphilic polar dyes for simultaneous 3D imaging of second harmonic generation (SHG) and two-photon excited fluorescence (TPF).
• Quantifying lipid ordering by normalizing SHG intensity to TPF intensity.
• Employing an enzyme release assay to detect membrane pore formation.
• Analyzing lipid dynamics, such as flip-flop, in wounded cell membranes.

Main Results:

• The SHG/TPF assay effectively assesses plasma membrane lipid ordering and reveals polycation effects at low concentrations.
• Polyethylenimine (PEI) reduced lipid order more significantly than poly-l-lysine (PLL).
• Poly-l-lysine (PLL) induced more pore formation than PEI.
• Lipid flip-flop dynamics were enhanced in the bleb membrane of wounded cells.
• The biocompatible polymer poly(N-(2-hydroxypropyl)methacrylamide) did not alter lipid order.

Conclusions:

• The developed SHG/TPF imaging technique provides a sensitive molecular-level assessment of plasma membrane structure.
• Polycations differentially affect membrane lipid ordering and integrity, with implications for biomaterial interactions.
• This method offers new insights into membrane dynamics and the effects of various agents on cell membranes.