Three‐Color Single‐Molecule Fluorescence Resonance Energy Transfer

A molecular ruler: A three-color single-molecule fluorescence resonance energy transfer (FRET) method is presented (see Figure inset), which can be used to simultaneously measure multiple molecular distance changes during molecular conformational changes and binding. The ability to directly study conformational subpopulations in a mixture of molecules with different interdye distances is highlighted by the well-separated peaks in the two- dimensional histogram in the Figure.