Recommended Dyes for dSTORM
dSTORM uses organic dyes, which can be switched by employing reducing agents in the buffer. Dyes with a high photon
yield per switching event, low on-off duty cycles, high survival fraction and a large number of switching cycles are
preferential. Many Xanthene, Coumarin and Cyanine derivatives fulfill these criteria. Companies often trade these under
special group names like Bodipy, Alexa Fluor (both from Invitrogen), Atto (from Sigma Aldrich), DyLight fluor (from
Thermo Scientific) and FluoProbes (from Interchim). In the literature, Alexa 647 has been mostly used as it has proved
to be a dye that matches all imaging criteria very well and can be switched easily between the dark and bright state.
The influence of the selected fluorophore has been nicely shown by G.T. Dempsey et al. (Evaluation of fluorophores
for optimal performance in localization-based super-resolution imaging, Nature Methods 2011). Please see below an
extract of the data from this publication.
Figure 4
(a-c): Effect of number of photons per on-switching event and the on-off duty cycle on STORM image quality for an example structure
(a ring-like object). (p, t and x): Images of CCPs (clathrin-coated pits) using Alexa Fluor 647 (p), Atto 655 (t) and Cy5.5 (x). Shown are composite
x–y cross-sections for ten CCPs aligned to their respective centers of mass along with the radial density distributions of localizations derived from
the composite x–y cross-sections. Scale bars: 100 nm.
For a multicolor experiment any combination between Atto 488, Cy3B/ Alexa 561 and Alexa 647/ DyLight 654
will work. Specifically the combination of Atto 488 with Alexa 647 has proven to be useful (see G.T. Dempsey et al.:
Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging, Nature Methods
2011) Recommendations for double labeling: Atto 488 + Atto 565 or Alexa 647
A nice option could also be: tdEOS/mEOS + Alexa647
Antibodies for dSTORM
If we consider the size of antibodies it is preferable to do direct antibody labeling without a primary and secondary
AB. Smaller antibodies such as nanobodies (cameloid like antibodies from camels, llamas and sharks) with a size in the
range of 2 nm may be preferred.
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