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The use of fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA‐sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well‐kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye‐target interactions, or to establish structural models of the dye‐target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous‐wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein‐attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single‐molecule FRET applications. Both pairs showed excellent performance in solution‐based smFRET experiments using alternating laser excitation. Minor differences in apparent dye‐protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF‐fluorophores are an attractive alternative to Alexa‐ and Cy‐dyes in smFRET studies or other fluorescence applications.

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series **