Biomineralization in diatoms is a highly complex process that occurs on many different length and time scales with the help of structure directing templates. During this process an intricate and extremely regular and reproducible nanoscale pattern of silica is produced through the interplay of specific organic molecules with silicic acid. Over the past years it has become evident that individual (bio)molecules cannot create the hierarchical silica nanopatterns. It was proposed that diatom biosilica-associated proteins (silaffins, silacidins, cingulins) in combination with long-chain polyamines (LCPA) assemble into nanopatterned templates that promote silica morphogenesis. To investigate this hypothesis we will address in this project two fundamental questions using single-molecule techniques: (i) What is the structural organization of the different cingulins in the nanopatterned, silica forming microrings of the diatom T. pseudonana? (ii) What are the interaction energies of scaffold proteins with silica, and between scaffold proteins? To study the first question, we will develop an assay based on super-resolution fluorescence microscopy to dissect the structural organization of cingulins within the insoluble organic matrix with nanometer resolution. In particular we will use the stochastic optical reconstruction microcopy and recently developed photo-switchable fluorescent proteins to either image extracted microrings or microrings within the intact diatom cell wall. The second question will be studied with a single-molecule force spectroscopy assay to probe molecular interactions between single proteins and polymers and silica surfaces. In particular, we will peel these biomolecules from silica surfaces to directly measure interaction forces and, by extension, interaction energies.