BALM: Watching the Formation of Tethered Bilayer Lipid Membranes with Submicron Lateral Resolution

Tethered bilayer lipid membranes (tBLMs) are artificial membranes largely used for the in situ study of biological membranes and membrane-associated proteins. To date, the formation of these membranes was essentially monitored by surface averaging techniques like surface plasmon resonance (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D), which cannot provide both local and real-time information in a single approach. Here, we report an original application of backside absorbing layer microscopy (BALM), a novel white-light wide-field optical microscopy, to study tBLMs. Thanks to the combination of sensitivity and resolution, BALM not only allowed the real-time quantitative monitoring of tBLM formation but also enabled the high-resolution visualization of local fluxes and matter exchanges taking place at each step of the process. Quantitative BALM measurements of the final layer thickness, reproduced in parallel with SPR, were consistent with the achievement of a continuous lipid bilayer. This finding was confirmed by BALM imaging, which additionally revealed the heterogeneity of the bilayer during its formation. While established real-time techniques, like SPR or QCM-D, view the surface as homogeneous, BALM showed the presence of surface patterns appearing in the first step of the tBLM formation process and governing subsequent matter adsorption or desorption steps. Finally, matter fluxes persisting even after rinsing at the end of the tBLM formation demonstrated the lasting presence of dispersed vesicular pockets with laterally fluctuating positions over the final single and continuous lipid bilayer. These new mechanistic insights into the tBLM formation process demonstrate the great potential of BALM in the study of complex biological systems.