In DNA nanotechnology, DNA is used as a structural material, rather than as an information carrier. The structural organization of the DNA itself determines accessibility to its underlying information content in vivo. Nucleosomes form the basic level of DNA compaction in eukaryotic nuclei. Nucleosomes sterically hinder enzymes that must bind the nucleosomal DNA, and hence play an important role in gene regulation. In order to understand how accessibility to nucleosomal DNA is regulated, it is necessary to resolve the molecular mechanisms underlying conformational changes in the nucleosome. Exploiting bottom-up control, we designed and constructed nucleosomes with fluorescent labels at strategically chosen locations to study nucleosome structure and dynamics in molecular detail with single-pair Fluorescence Resonance Energy Transfer (spFRET) microscopy. Using widefield total internal reflection fluorescence (TIRF) microscopy on immobilized molecules, we observed and quantified DNA breathing dynamics on individual nucleosomes. Alternatively, fluorescence microscopy on freely diffusing molecules in a confocal detection volume allows a fast characterization of nucleosome conformational distributions.