Biotium, a leading life science reagent supplier and producer of innovative dyes, has collaborated with Professor Ke Xu at University of California, Berkeley to generate a strategy for improving the photoswitching behaviour of rhodamine dyes for optimal (d)STORM [(direct) stochastic optical reconstruction microscopy] performance. The two rhodamine-based and green-excitable CF dyes produced, CF583R and CF597R, display significant sensitisation toward photoswitching and offer comparable performance to the commonly used red-excited Alexa Fluor 647 dye, making high-quality two-colour three-dimensional (3D) imaging possible even for difficult structures.
Identifying and developing effective photoswitchable dyes is central to (d)STORM. Though there are several dyes (e.g., Alexa Fluor 647, CF660C, and CF680) excited by red lasers that perform suitably for (d)STORM, generating comparable dyes in other color channels remains a challenge. In a recent Angewandte Chemie publication, scientists at Biotium designed a new chemical strategy for generating these elusive dyes. The breakthrough was achieved by replacing the benzene ring in the cores of rhodamine-based dyes with the permanently charged 1,3-disubstituted imidazolium to create CF583R and CF597R. The positively charged 1,3-disubstituted imidazolium sensitises the dye for the photoswitching process under (d)STORM conditions by facilitating electron capture in a thiol-containing reductive buffer and stabilisation by a resonance structure. Under green excitation, CF583R and CF597R exhibited outstanding (d)STORM performance with fast on-off switching, long-lasting blinking, and bright single-molecule emission. The authors found that other green-excited (d)STORM dyes (i.e., Alexa Fluor 532, Cy3B, Atto 565, and Alexa Fluor 568) exhibited very limited photoswitching under the same excitation power, with the latter two yielding no discernible spectral changes. In this recent Angewandte Chemie publication, the authors, Wang et al., demonstrated the use of CF583R and CF597R in groundbreaking high-quality two-colour 3D-(d)STORM and excellent quality imaging of the challenging structure of actin cytoskeletons.
“Working closely with Biotium chemists and communicating the ideal chemical properties needed for optimal STORM dyes, our collaboration was able to yield a framework for developing more fluorescent colours that expand the colour palette for multicolour STORM imaging” says Ke Xu PhD, Associate Professor of Chemistry at UC Berkeley.
"While there have been recent significant advancements in STORM imaging and applications, the work toward improving the chemical probes for green-excited dyes for the 532-561 nm lasers has been lacking” says Wai-Yee Leung PhD, Vice President of Research and Development at Biotium. “We are proud to offer novel reagents for multiplex imaging to support this exciting field.”