Zeiss ELYRA - Recommended Dyes for dSTORM; Dyes for dSTORM; Antibodies for dSTORM

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fulllthesecriteria.Companiesoftentradetheseunder
specialgroupnameslikeBodipy,AlexaFluor(bothfromInvitrogen),Atto(fromSigmaAldrich),DyLightuor(from
ThermoScientic)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inuenceoftheselecteduorophorehasbeennicelyshownbyG.T.Dempseyetal.(Evaluationofuorophores
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.SpecicallythecombinationofAtto488withAlexa647hasproventobeuseful(seeG.T.Dempseyetal.:
Evaluationofuorophores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.
White Paper
7