Common to these short-range methods is their reliance on site-specific labeling with synthetic fluorophores, which is best outlined using the example of FRET: By non-radiative energy transfer through dipole-dipole interactions, FRET measures distances between a donor and an acceptor fluorophore in the range of 3–10 nm. The requirements for the FRET probes used on a single molecule level are: brightness, photostability, and the formation of a FRET pair between the two probes. FRET pairing requires that the emission spectrum of the energy donor has sufficient overlap with the excitation spectrum of the acceptor. In fact, the proper choice of dye pair also tunes the length scale where the measurement is most sensitive, depending on the degree of spectral overlap between the two fluorophores 69. Cy3/Cy5 are a frequently used FRET pair 70–72, as well as Alexa488/Alexa594 32, 33, 73, 74. Orange dyes such as Alexa 555 75, TAMRA, and a few variants of Atto, are also used by many. Red dyes, such as Atto647N and Alexa647 are popular alternatives to Cy5 31, 69. FRET determination experiments were initially based on fluorescence intensities, but it has become clear that this method suffers from many artifacts. Limited rotational freedom, for instance, can affect FRET 64, 76, and distance determination is highly sensitive to background fluorescence, cross-contamination between the different detection channels, and other quenching mechanisms. For these reasons, FRET is mainly used to detect relative changes. However, multiparameter readouts analyzing fluorescence anisotropy, intensity, and lifetime are also effective at providing reliable distance readouts 32, 33, 77, 78. Consequently, dyes with longer lifetimes are better suited for single molecule studies, and unfortunately many red dyes suffer from short lifetimes 79. Also, multi-color applications have been developed and are increasingly used. However, these applications so far only work with intensity-based FRET, and because of their complexity, they are mainly used to measure relative distance changes 80–84. To avoid labeling with, and complex detection of, multiple colors, a multi-acceptor labeling strategy has been developed based on reversible photoswitching of the acceptor dye, similar to dSTORM, so that only one distance can be probed at a time 72.