# Why Do Tethered-Bilayer Lipid Membranes Suit for Functional Membrane Protein Reincorporation?
## 3 Characterization of Tethered-Bilayer Lipid Membranes  
Because they are firmly held in place, tBLMs are considerably more robust than supported lipid bilayers such as black or bilayer lipid membranes (BLMs) [79; 80], also renamed free-standing lipid layer or suspended-lipid bilayers, which are originally formed across on a microsized-aperture, and more recently, on nanopores [26; 27; 28]. Generally speaking, tBLMs typically show a high robustness and long-term stability and hence, they are accessible to a portfolio of different analytical tools operating at a bulk interface [42]. They include imaging techniques, like _atomic force microscopy_ (AFM) [30; 81; 82; 83; 84; 85; 86; 87] and _fluorescence microscopy_ (FM) [34; 87; 88; 89], _fluorescence recovering after photobleaching_ (FRAP) [35; 81; 87; 90; 91; 92; 93], _neutron reflectometry_ (NR) [79; 94; 95; 96; 97; 98; 99; 100; 101] and _X-ray photoelectron spectroscopy_ (XPS) [20; 82; 86; 87; 102; 103], spectroscopic techniques such as _ellipsometry_[94; 104], _infrared reflection absorption spectroscopy_ (IRRAS) [96; 105; 106] or _surface-enhanced infrared absorption spectroscopy_ (SEIRAS) [107; 108; 109; 110], _surface plasmon resonance_ (SPR) [30; 34; 35; 37; 39; 111; 112; 113] or _quartz crystal micro-balance with dissipation monitoring_ (QCM-D) [30; 39; 85; 112], as well as electrochemical methods such as _electrochemical impedance spectroscopy_ (EIS) [114; 115; 116; 117; 118; 94; 108; 109; 117; 118; 119; 120; 121; 122; 123] and current-voltage (CV) analysis [118; 119; 120; 121; 122; 123]. These techniques are sensitive to net changes in packing or interfacial mass (QCM-D or ellipsometry), bilayer morphology (AFM, FM), the presence of chemical groups (IR, XPS), structure and composition (NR) have been used so far to evaluate the full picture of the lipid membranes (i.e., structure, composition and functional properties) and represent a very powerful combination to unravel the mechanism of biomolecular interactions. While SPR and QCM-D allow real-time monitoring of the tBLM formation in a label-free format, fluorescence microscopy (FM) and FRAP investigate domain morphology and membrane dynamics with the measurement of the lateral diffusion of lipids, respectively. AFM has been used to gather surface details in terms of occurrence of peculiar structure and defects. One of the unique features of this latter technique is that it can measure surface forces with a nanometer lateral resolution. Recently, single-molecule force spectroscopy (FS) measurements have provided in-depth insight to assess the orientation of reconstituted transmembrane proteins in tBLMs [86]. NR also provides high resolution structural information on lipid bilayer stacking and internal distribution of components after interaction between intrinsic proteins and disordered membrane [124]. EIS is an excellent tool to characterize the electric properties of membrane including resistance and capacitance. This large panel allows a fine characterization of tBLMs during and after their formation, in terms of structure, (optical or acoustic) thickness, fluidity and sealing [125]. It shows that the chemical nature of the sub-membrane space has a significant impact on both the structure of the lipid bilayer and the functional incorporation of membrane components [51]. The possible combination of multiple complementary measurements with biologically accurate samples is key for a realistic understanding of membrane related phenomena. Only through the use of complementary techniques, such as the ones hereby mentioned, does it become a realistic aim to resolve the relative position, orientation and distribution of the membrane components to obtain detailed information on molecular mechanisms by which peptides, proteins or other chemical compounds (e.g., drugs) interact with biomembranes. Table 1, adapted from [PERSON] and [PERSON] [126], [PERSON] et al. [70], [PERSON] et al.