Sednev Maksim V, Belov Vladimir N, Hell Stefan W
Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Methods Appl Fluoresc. 2015 Oct 22;3(4):042004. doi: 10.1088/2050-6120/3/4/042004.
The review deals with commercially available organic dyes possessing large Stokes shifts and their applications as fluorescent labels in optical microscopy based on stimulated emission depletion (STED). STED microscopy breaks Abbe's diffraction barrier and provides optical resolution beyond the diffraction limit. STED microscopy is non-invasive and requires photostable fluorescent markers attached to biomolecules or other objects of interest. Up to now, in most biology-related STED experiments, bright and photoresistant dyes with small Stokes shifts of 20-40 nm were used. The rapid progress in STED microscopy showed that organic fluorophores possessing large Stokes shifts are indispensable in multi-color super-resolution techniques. The ultimate result of the imaging relies on the optimal combination of a dye, the bio-conjugation procedure and the performance of the optical microscope. Modern bioconjugation methods, basics of STED microscopy, as well as structures and spectral properties of the presently available fluorescent markers are reviewed and discussed. In particular, the spectral properties of the commercial dyes are tabulated and correlated with the available depletion wavelengths found in STED microscopes produced by LEICA Microsytems, Abberior Instruments and Picoquant GmbH.
本综述涉及具有大斯托克斯位移的市售有机染料及其作为基于受激发射损耗(STED)的光学显微镜中的荧光标记物的应用。STED显微镜突破了阿贝衍射极限,提供了超越衍射极限的光学分辨率。STED显微镜是非侵入性的,需要将光稳定的荧光标记物附着到生物分子或其他感兴趣的物体上。到目前为止,在大多数与生物学相关的STED实验中,使用的是斯托克斯位移为20 - 40纳米的明亮且耐光的染料。STED显微镜的快速发展表明,具有大斯托克斯位移的有机荧光团在多色超分辨率技术中是不可或缺的。成像的最终结果依赖于染料、生物共轭过程和光学显微镜性能的最佳组合。本文对现代生物共轭方法、STED显微镜的基础知识以及目前可用的荧光标记物的结构和光谱特性进行了综述和讨论。特别是,将商业染料的光谱特性制成表格,并与徕卡显微系统公司、阿贝里奥仪器公司和皮可量子有限公司生产的STED显微镜中可用的损耗波长相关联。