Content area
Full Text
ANALYSIS
Evaluation of uorophores for optimal performance in localization-based super-resolution imaging
2011Nature America, Inc. All rights reserved.
One approach to super-resolution uorescence imaging uses sequential activation and localization of individual uorophores to achieve high spatial resolution. Essential to this techniqueis the choice of uorescent 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 identied several photoswitchable dyes with good to excellent performance in four independent spectral ranges, with which we demonstrated lowcross-talk, four-color super-resolution imaging.
The spatial resolution limit of light microscopy imposed by diffraction has recently been overcome by super-resolution fluorescence imaging methods1,2. Among these methods, stochastic optical reconstruction microscopy (STORM)3 and (fluorescence) photoactivated localization microscopy4,5 can
be used to achieve sub-diffraction-limit resolution by sequentially imaging and localizing individual fluorophores that label the target structure. The basic concept of single-molecule-localizationbased super-resolution imaging entails (i) sparse activation of an optically resolvable subset of fluorophores, (ii) determination of the positions of these fluorophores with sub-diffraction-limit precision and (iii) repeating this process to allow stochastically different subsets of fluorophores to be turned on and localized each time (Fig. 1a). An image with subdiffraction-limit resolution can then be reconstructed from the positions of the many activated molecules.
As stated in the original STORM paper3, we demonstrate the concept of STORM using the cyanine switch, but in principle any suitable optically switched fluorophore could be used and the
Graham T Dempsey1,6, Joshua C Vaughan2,3,6, Kok Hao Chen3,6, Mark Bates4 & Xiaowei Zhuang2,3,5
STORM concept is also applicable to other photoswitchable fluorophores and fluorescent proteins. Indeed, a variety of fluorescent probes have been used for localization-based super-resolution imaging, including organic dyes3,621, fluorescent proteins4,5,22,23 and quantum dots24. In the simplest imaging mode, continuous illumination of a single dye (for example, Alexa...