title: Multi-colour direct STORM with red emitting carbocyanines.
abstract: BACKGROUND
  Single molecule-based super-resolution methods have become important tools to study nanoscale structures in cell biology. However, the complexity of multi-colour applications has prevented them from being widely used amongst biologists. Direct stochastic optical reconstruction microscopy (dSTORM) offers a simple way to perform single molecule super-resolution imaging without the need for an activator fluorophore and compatible with many conventionally used fluorophores. The search for the ideal dye pairs suitable for dual-colour dSTORM has been compromised by the fact that fluorophores spectrally apt for dual-colour imaging differ with respect to the optimal buffer conditions required for photoswitching and the generation of prolonged non-fluorescent (OFF) states.
  
  
  RESULTS
  We present a novel variant of dSTORM that combines advantages of spectral demixing with the buffer compatible blinking properties of red emitting carbocyanine dyes, spectral demixing dSTORM (SD-dSTORM). In contrast to previously published work, SD-dSTORM requires reduced laser power and fewer imaging frames for the faithful reconstruction of super-resolved biological nanostructures. In addition, SD-dSTORM allows the use of commercially available rather than custom-made probes and does not rely on potentially error-prone cross-talk correction, thus allowing reliable co-localisation.
  
  
  CONCLUSIONS
  SD-dSTORM presents a significant advance towards user-friendly single molecule localisation-based super-resolution microscopy combining advantages of state-of-the-art methodologies to perform fast, reliable and efficient multi-colour dSTORM.
authors.affiliation: Institute of Chemistry, Freie Universität Berlin, 14195 Berlin, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Lampe
authors.forename: Volker
authors.initials: V
authors.lastname: Haucke
authors.forename: Stephan J
authors.initials: SJ
authors.lastname: Sigrist
authors.forename: Mike
authors.initials: M
authors.lastname: Heilemann
authors.forename: Jan
authors.initials: J
authors.lastname: Schmoranzer
mesh_terms: D000465:Algorithms; D000818:Animals; D002232:Carbocyanines / Q000737:chemistry*; D002460:Cell Line; D002522:Chlorocebus aethiops; D003116:Color; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry*; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D007834:Lasers; D008027:Light; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D008870:Microtubules / Q000648:ultrastructure*
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes