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.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.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.
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