Only 15 years ago macromolecular crowding was still underappreciated. The last years have seen an increasing relevant number of crowding-regulated mechanisms coming to light; as the regulation of gene expression, the gating energies of membrane proteins or the regulation of the membrane protein conformation landscape.

The cell membrane is crowded with proteins, protein content reaches area fractions of 0.55 and even crystalline densities in extreme cases.

High Speed Atomic Force Microsocopy has brought new possibilities to study macromolecular crowding in membranes, providing location and structural information on unlabelled molecules with sub-second temporal resolution and full visualization of the local crowding of the molecular nanoenvironment.

The voltage-dependent anion channel (VDAC) is the most abundant protein of the mitochondrial outer membrane, mediating the exchange of ions and metabolites across the membrane. Beyond this fundamental role, VDAC regulates key mitochondrial functions—including apoptosis, metabolism, and the release of mitochondrial nucleic acids into the cytosol—positioning it as a central hub in cellular regulation. VDAC oligomerization has been implicated in several key physiological and pathological processes, such as mitochondrial DNA release, calcium homeostasis, or diabetes, lipid scrambling , and mitochondrial organization . However, how distinct VDAC assemblies contribute to these processes remains unresolved Importantly, VDAC does not form uniform, well-defined oligomers, but rather heterogeneous lipid–VDAC assemblies that adopt honeycomb-like topologies, which we recently characterized. The compaction of these assemblies is tightly regulated by cholesterol levels and likely plays a crucial role in mitochondrial physiology. 

However, their structural heterogeneity and the high abundance of VDAC in the MOM make them difficult to resolve using conventional low-resolution techniques such as cross-linking, proximity ligation, fluorescence microscopy, or FRET. Atomic Force Microscopy (AFM) offers 1–2 nm spatial resolution—surpassing optical super-resolution methods (20–50 nm)—and enables label-free visualization of individual membrane proteins and their organization within native membranes. Furthermore, high-speed AFM (HS-AFM) extends these capabilities to the temporal domain, capturing dynamic molecular processes at over 10 frames per second while preserving nanoscale detail. 

The lateral organization of bacterial photosynthetic membranes is particularly important for their function because (i) the light transfer from light harvesting proteins to the reaction center depends on protein organization [1], and (ii) regulates the diffusion of quinone from the reaction center to the cytochrome bc1 complex.

Thanks to AFM we visualize and analyze the protein organization and function of bacterial photosynthetic membranes, this information is next combined with an optical spectroscopy photosynthetic energy flow assay to correlate funtion with the photosynthetic membrane supramolecular organisation. Moreover, the use of nanoscale engineering of the supramolecular organisation architecture entails a better comprehension of the photosynthetic membrane functioning.

The study of the mechanical properties of viruses has become fundamental in physical virology. It is crucial not only to know the structure of viral-nucleic acid complexes, but also the physical properties at the nanoscale such as the mechanical stability, elasticity, and dynamical responses of viruses to changes in the environmental conditions, lile pH, and to the interaction with target molecules like the host cell membranes. Only the Atomic Force Microscopy can provide this sort of information. Structural Biology techniques such as Electron-microscopy or X-Ray diffraction cannot assess this information.