Direct observation of thermally induced fluctuation in lipid membranes using TEM-based gold nanoparticle tracking

Aim: This study aims to develop and validate a transmission electron microscopy (TEM)–based approach for probing nanoscale lipid membrane dynamics by tracking the motion of gold nanoparticles dispersed on membrane surfaces. Methods: Lipid thin films composed of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) were prepared over 2 μm holes in Quantifoil grids, and 5 nm gold nanocolloids were introduced as tracer particles. Sequential TEM imaging was performed during controlled heating and cooling cycles, and nanoparticle trajectories were analyzed to obtain mean squared displacement (MSD) curves. These measurements enabled quantification of thermally driven membrane dynamics. The temperature dependent behavior was further compared with differential scanning calorimetry (DSC) of dehydrated lipid samples. Results: DPPC exhibited a pronounced MSD peak near 52.5 °C during the first heating cycle, corresponding to its main phase transition, whereas DOPC showed gradual and continuous mobility changes consistent with its intrinsically disordered acyl chains. Differences between electron beam molecular dynamics (EBMD) and DSC transition temperatures likely arose from dehydration and thin film geometry. Across repeated thermal cycles, DPPC membranes displayed cycle dependent changes in MSD profiles, suggesting annealing induced homogenization and potential beam induced structural alterations. Conclusions: EBMD provides real space, time resolved visualization of nanoscale membrane fluctuations and complements ensemble techniques such as DSC, fluorescence recovery after photobleaching (FRAP), and nuclear magnetic resonance (NMR). The TEM based particle tracking approach reliably distinguishes ordered versus disordered lipid systems and offers a versatile platform for investigating soft biological membranes, including systems containing proteins or heterogeneous lipid compositions.