Multi-colour direct STORM with red emitting carbocyanines
Multi-colour direct STORM with red emitting carbocyanines
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DOI：
10.1111/boc.201100011
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发表时间：
2012-04-01
影响因子：
2.7
通讯作者：
Schmoranzer, Jan
中科院分区：
文献类型：
--
作者：
Lampe, Andre;Haucke, Volker;Schmoranzer, Jan
背景资料。基于单分子的超分辨方法已经成为研究细胞生物学中纳米结构的重要工具。然而，多色应用的复杂性阻止了它们在生物学家中的广泛应用。直接随机光学重建显微镜（dSTORM）提供了一种简单的方法来执行单分子超分辨率成像，而不需要激活剂荧光团，并与许多常规使用的荧光团兼容。寻找适合双色dSTORM的理想染料对已经受到这样一个事实的影响，即光谱上适合双色成像的荧光团在光开关所需的最佳缓冲条件和长时间非荧光（OFF）状态的产生方面有所不同。我们提出了一种新的变体dSTORM，结合了光谱分层与缓冲液兼容的闪烁性能的红色发光碳菁染料，光谱分层dSTORM（SD-dSTORM）的优势。与以前发表的工作相比，SD-dSTORM需要更低的激光功率和更少的成像帧来忠实地重建超分辨生物纳米结构。此外，SD-dSTORM允许使用市售探针而非定制探针，并且不依赖于潜在的易错串扰校正，从而实现可靠的共定位。SD-dSTORM在用户友好的基于单分子定位的超分辨率显微镜方面取得了重大进展，结合了最先进方法的优势，可快速，可靠和高效地执行多色dSTORM。
Background information. 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.