Randall Centre for Cell and Molecular Biophysics, King's College London, New Hunt's House, Guy's Campus, London SE1 1UL, United Kingdom.
Methods Appl Fluoresc. 2018 Jul 9;6(4):045002. doi: 10.1088/2050-6120/aad018.
Superresolution microscopy based on localisation is usually performed in a buffer containing enzymatic oxygen scavenger, which facilitates reversible photoswitching of the dye molecules. This makes correlative fluorescence localisation and atomic force microscopy (AFM) challenging, because enzymatic oxygen scavenging interferes with the AFM cantilevers. Here we report on the blinking kinetics of a new red cyanine dye, iFluor-647, which is similar to the Alexa-647 dye commonly used for superresolution microscopy, but with brightness and blinking properties which are superior to Alexa-647 in a buffer without enzymatic oxygen scavenger. We measure the blinking behaviour of iFluor-647 in buffers with and without enzymatic oxygen scavenger with different thiol concentrations. We then apply this dye for correlative localisation and atomic force microscopy in a buffer without enzymatic oxygen scavenger, which allows acquisition of AFM and superresolution images without buffer change.
基于定位的超分辨率显微镜通常在含有酶氧清除剂的缓冲液中进行，这有利于染料分子的可逆光开关。这使得相关的荧光定位和原子力显微镜（AFM）变得具有挑战性，因为酶氧清除剂会干扰 AFM 悬臂。在这里，我们报告了一种新型红色菁染料 iFluor-647 的闪烁动力学，它类似于常用于超分辨率显微镜的 Alexa-647 染料，但在没有酶氧清除剂的缓冲液中，其亮度和闪烁特性优于 Alexa-647。我们在有和没有酶氧清除剂的缓冲液中测量了 iFluor-647 的闪烁行为，缓冲液中含有不同浓度的硫醇。然后，我们在没有酶氧清除剂的缓冲液中应用这种染料进行相关的定位和原子力显微镜，这允许在不更换缓冲液的情况下获取 AFM 和超分辨率图像。
