Support & Resources


UC Berkeley and Biotium identify dye modifications for superior fluorescence photoswitching in STORM

Super-resolution microscopy is an exciting new field for fluorescence microscopy that overcomes the barriers for image resolution imposed by the diffraction limit of light. Stochastic optical reconstruction microscopy (STORM) is a type of super-resolution imaging that uses stochastic on-off switching of single-molecule fluorescence to localize millions of molecules over many camera frames to produce images with ~10 nm spatial resolutions. Direct stochastic optical reconstruction microscopy (dSTORM) methods, based on photoswitching of synthetic dyes, have high single-molecule brightness and resolution. However, high-quality multicolor dSTORM remains difficult due to the lack of ideal dyes outside of the red excitation range (e.g., 633 or 640 nm).

In a recent publication in Angewandte Chemie, B. Wang et al. from UC Berkeley, in collaboration with Biotium scientists, proposed a strategy to improve the photoswitching behavior of rhodamine dyes, leading to the development of two green-excitable dyes for high quality multi-color STORM imaging. The photoswitching mechanism of rhodamine dyes can be induced under reducing conditions to generate a long-lived photoactivated radical dark state, which reverts to a fluorescence emitting state with violet light or the addition of oxygen. Using this knowledge, the authors successfully developed two rhodamine-based dyes, CF®583R and CF®597R, by replacing the benzene ring in the rhodamine core with a positively charged 1,3-disubstituted imidazolium, which would further stabilize the radical through a resonance structure. Experiments done with these dyes showed fast on-off switching, long lasting blinking, and bright single-molecule emission, characteristics of high performance dSTORM dyes. The novel, green-excited dyes demonstrated similar results to validated, red-excited dyes in the same imaging buffer.

The authors compared the spectral behavior of CF®583R and CF®597R with other green-excited dSTORM dyes, such as Alexa Fluor® 532, Cy®3B, Atto 565, and Alexa Fluor® 568. These older generation dyes showed very limited photoswitching, while CF®583R and CF®597R showed substantially higher rates of photoswitching. CF®583R and CF®597R both yielded high-resolution images, with better uniformity and continuity than the other green-excited dyes tested. Based on these results, the authors concluded that replacing the benzene ring with a positively charged imidazolium sensitized rhodamine-based dyes towards photoswitching in the dSTORM buffer with improved imaging results. The images produced using this protocol were high quality and on par with those obtained using red-excited dyes. This chemical strategy can therefore be used to develop other fluorescent dyes for STORM imaging, advancing potential for multi-color super-resolution microscopy.

Comparison of typical (d)STORM images obtained in fixed COS-7 cells for microtubules immunolabeled by different dyes. (d)STORM was performed under standard conditions using a standard tris-based (d)STORM imaging buffer containing 100 mM cysteamine and an oxygen scavenger. (a) CF®583R. (b) CF®597R. (c) Alexa Fluor® 532. (d) Cy3B. (e) CF®568. Localization distributions are further given for single CF®583R and CF®597R molecules in the sample, in the X (in-plane; top) and Z (depth; bottom) directions, respectively. Gaussian fits (red curves) give standard deviations of ~10 and ~20 nm in the two directions, respectively. Color denotes depth (Z) values. Images and figures courtesy of Bowen Wang, Michael Xiong, and Professor Ke Xu, College of Chemistry, University of California, Berkeley.

Learn more about Biotium’s CF® Dyes, and their unrivaled performance for super-resolution applications.

Full Citation:

Wang, B., Xiong, M., Susanto, J., Li, X., Leung, W. Y., & Xu, K. (2021). Transforming Rhodamine Dyes for (d)STORM Super‐Resolution Microscopy via 1,3‐Disubstituted Imidazolium Substitution. Angewandte Chemie.