Biotium has collaborated with Professor Ke Xu at the University of California, Berkeley, in order to improve 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. Although there are several dyes (eg, Alexa Fluor 647, CF660C and CF680) excited by red lasers that perform suitably for (d)STORM, generating comparable dyes in other colour 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 (ie, 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 cytoskeleton.
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