An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging.
An orange fluorescent protein with a large Stokes shift for single-excitation multicolor FCCS and FRET imaging.
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DOI：
10.1021/ja3018972
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发表时间：
2012-05-09
影响因子：
15
通讯作者：
Verkhusha, Vladislav V.
中科院分区：
文献类型：
--
作者：
Shcherbakova, Daria M.;Hink, Mark A.;Joosen, Linda;Gadella, Theodorus W. J.;Verkhusha, Vladislav V.
基于基因编码荧光蛋白（FP）的多色成像是研究活细胞中几个动态过程的有力方法。我们报告了一个单体橙子FP与一个大的斯托克斯位移（LSS），称为LSSm橙子（激发/发射在437/572 nm），它填补了现有的光谱间隙之间的绿黄色和红色LSSFP。LSSmOrange的亮度比最亮的红色LSSFP的亮度大5倍，并且类似于绿黄色LSS-FP。LSSmOrange允许使用单一激发波长的多种荧光应用，这在以前是不可能的。使用LSSmOrange，我们开发了一个四色单激光荧光互相关光谱，完全基于FP。四重互相关结合光子计数直方图技术允许定量单分子分析的颗粒标记有四个FP。LSSmOrange进一步应用于同时成像两个Förster共振能量转移对，其中一个是常用的CFP-YFP对，具有单个激发激光。LSSmOrange-mKate 2和CFP-YFP生物传感器的组合能够真实的实时成像凋亡活性和钙波动。LSSmOrange诱变、低温和同位素效应研究揭示了负责LSS表型的激发态质子转移的质子中继。
Multicolor imaging based on genetically-encoded fluorescent proteins (FPs) is a powerful approach to study several dynamic processes in a live cell. We report a monomeric orange FP with a large Stokes shift (LSS), called LSSmOrange (excitation/emission at 437/572 nm), which fills up an existing spectral gap between the green-yellow and red LSSFPs. Brightness of LSSmOrange is 5-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSS-FPs. LSSmOrange allows numerous multicolor applications using a single excitation wavelength that was not possible before. Using LSSmOrange we developed a four-color single-laser fluorescence cross-correlation spectroscopy, solely based on FPs. The quadruple cross-correlation combined with photon counting histogram techniques allowed quantitative single-molecule analysis of the particles labeled with four FPs. LSSmOrange was further applied to simultaneously image two Förster resonance energy transfer pairs, one of which is the commonly used CFP-YFP pair, with a single excitation laser. The combination of LSSmOrange-mKate2 and CFP-YFP biosensors enabled imaging of apoptotic activity and calcium fluctuations in real time. The LSSmOrange mutagenesis, low-temperature and isotope effect studies revealed a proton relay for the excited state proton transfer responsible for the LSS phenotype.
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DOI：
10.1073/pnas.180317197
发表时间：
2000-09-12
影响因子：
11.1
作者：
Heinze, KG;Koltermann, A;Schwille, P
通讯作者：
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影响因子：
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影响因子：
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