Membrane proteins play crucial roles in various cellular processes, making them key targets for drugs. Traditional methods often rely on purified proteins or reconstitution in artificial lipid environments. However, reconstituted systems may not accurately replicate the complex heterogeneity of the native membranes, and how this affects protein structure and dynamics is often elusive. In recent years, several approaches have been developed to observe outer membrane proteins in their native surroundings. Selective observation of -helical membrane proteins in their native surroundings is still a major challenge for highresolution techniques such as X-ray crystallography or cryo-EM. On the other hand, spectroscopic observations are challenged by the low expression level and difficulty of selective labeling. Solidstate nuclear magnetic resonance spectroscopy has made significant progress in this direction. Site-directed spin labeling combined with electron spin resonance (ESR) spectroscopy is a complementary technique for investigating the conformation and heterogeneity of biomolecules, even within their native contexts. Pulsed ESR spectroscopy techniques, including pulsed electronelectron double resonance (PELDOR or DEER) is ideally suited for such experiments due to their high sensitivity, even at nanomolar concentrations. Over the past few years, we demonstrated that PELDOR can be used to observe the outer membrane proteins (OMPs) of E. coli in the isolated membranes. In Gram-negative bacteria such as E. coli, the cellular envelope consists of an outer membrane (OM) and an inner membrane (IM) separated by the periplasm. Here, the inner membrane was selectively solubilized, which, combined with the natural cysteineexclusion in OMPs, enabled selective labeling and distance measurements using nitroxide, Gd 3+, and trityl labels in the isolated outer membranes. However, direct spin labeling and distance determination in the inner membrane (IM) have not yet been demonstrated for helical inner membrane proteins (IMPs). Spin-labeled nanobodies were employed to probe IMP conformation in the native envelope. However, the general application of this approach is severely limited due to the requirement for conformationspecific nanobodies. Unlike the OM, numerous native cysteines of IMPs hinder conventional labeling and PELDOR experiments employing nitroxide labels. In addition to the effect of the native lipids and other interacting partners, the role of nanoconfinement (within the OM-periplasm-IM chamber) on the conformation and interaction of IMPs remains largely unknown.