Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging

• Published in: Nature methods Vol. 8; no. 12; pp. 1027 - 1036
• Main Authors: Dempsey, Graham T, Vaughan, Joshua C, Chen, Kok Hao, Bates, Mark, Zhuang, Xiaowei
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-12-2011; Nature Publishing Group
• Subjects: 631/1647/1888/1493; 631/1647/245/2225; 631/1647/328/2238; 639/638/439/945; analysis; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Buffers; Color; Diagnostic imaging; Dyes; Fluorescence; Fluorescent Dyes - analysis; Life Sciences; Light; Luminescent Measurements; Microscopy; Microscopy, Fluorescence; Microtubules - metabolism; Molecular Imaging - methods; Molecular Probes - analysis; Molecular Probes - chemistry; Particle Size; Photons; Probes; Proteomics; Scientific imaging; United States
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth.1768

A quantitative characterization of the switching properties of 26 organic dyes relates these properties to the quality of localization-based super-resolution images they generate. The data are a useful resource for selecting dyes and point to avenues for future analysis.
One approach to super-resolution fluorescence imaging uses sequential activation and localization of individual fluorophores to achieve high spatial resolution. Essential to this technique is the choice of fluorescent probes; the properties of the probes, including photons per switching event, on-off duty cycle, photostability and number of switching cycles, largely dictate the quality of super-resolution images. Although many probes have been reported, a systematic characterization of the properties of these probes and their impact on super-resolution image quality has been described in only a few cases. Here we quantitatively characterized the switching properties of 26 organic dyes and directly related these properties to the quality of super-resolution images. This analysis provides guidelines for characterization of super-resolution probes and a resource for selecting probes based on performance. Our evaluation identified several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated low–cross-talk, four-color super-resolution imaging.