Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
J Am Chem Soc. 2012 May 9;134(18):7913-23. doi: 10.1021/ja3018972. Epub 2012 Apr 24.
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 five-fold larger than that of the brightest red LSSFP and similar to the green-yellow LSSFPs. LSSmOrange allows numerous multicolor applications using a single-excitation wavelength that was not possible before. Using LSSmOrange we developed 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 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 line. 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.
基于遗传编码荧光蛋白（FPs）的多色成像技术是研究活细胞中多个动态过程的有力方法。我们报告了一种单体橙色 FP，具有较大的斯托克斯位移（LSS），称为 LSSmOrange（激发/发射波长为 437/572nm），它填补了绿黄色和红色 LSSFPs 之间现有的光谱间隙。LSSmOrange 的亮度比最亮的红色 LSSFP 大五倍，与绿黄色 LSSFPs 相似。LSSmOrange 允许使用以前不可能的单一激发波长进行多种多色应用。使用 LSSmOrange，我们开发了仅基于 FPs 的四色单激光荧光互相关光谱法。四重互相关结合光子计数直方图技术允许对用四个 FP 标记的粒子进行定量单分子分析。LSSmOrange 进一步用于同时用单激发激光线成像两个Förster 共振能量转移对，其中一个是常用的 CFP-YFP 对。LSSmOrange-mKate2 和 CFP-YFP 生物传感器的组合能够实时成像凋亡活性和钙波动。LSSmOrange 的诱变、低温和同位素效应研究揭示了激发态质子转移的质子传递，这是 LSS 表型的原因。
