Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany.
Biophys J. 2013 Aug 20;105(4):848-61. doi: 10.1016/j.bpj.2013.05.059.
Fluorescence fluctuation imaging is a powerful means to investigate dynamics, interactions, and stoichiometry of proteins inside living cells. Pulsed interleaved excitation (PIE) is the method of nanosecond alternating excitation with time-resolved detection and allows accurate, independent, and quasi-simultaneous determination of fluorescence intensities and lifetimes of different fluorophores. In this work, we combine pulsed interleaved excitation with fluctuation imaging methods (PIE-FI) such as raster image correlation spectroscopy (RICS) or number and brightness analysis (N&B). More specifically, we show that quantitative measurements of diffusion and molecular brightness of Venus fluorescent protein (FP) can be performed in solution with PIE-RICS and compare PIE-RICS with single-point PIE-FCS measurements. We discuss the advantages of cross-talk free dual-color PIE-RICS and illustrate its proficiency by quantitatively comparing two commonly used FP pairs for dual-color microscopy, eGFP/mCherry and mVenus/mCherry. For N&B analysis, we implement dead-time correction to the PIE-FI data analysis to allow accurate molecular brightness determination with PIE-NB. We then use PIE-NB to investigate the effect of eGFP tandem oligomerization on the intracellular maturation efficiency of the fluorophore. Finally, we explore the possibilities of using the available fluorescence lifetime information in PIE-FI experiments. We perform lifetime-based weighting of confocal images, allowing us to quantitatively determine molecular concentrations from 100 nM down to <30 pM with PIE-raster lifetime image correlation spectroscopy (RLICS). We use the fluorescence lifetime information to perform a robust dual-color lifetime-based FRET analysis of tandem fluorescent protein dimers. Lastly, we investigate the use of dual-color RLICS to resolve codiffusing FRET species from non-FRET species in cells. The enhanced capabilities and quantitative results provided by PIE-FI make it a powerful method that is broadly applicable to a large number of interesting biophysical studies.
荧光波动成像技术是一种强大的方法，可用于研究活细胞内蛋白质的动力学、相互作用和化学计量。脉冲交错激发（PIE）是一种纳秒交替激发方法，结合时间分辨检测，可实现不同荧光团荧光强度和寿命的精确、独立和准同时测定。在这项工作中，我们将脉冲交错激发与波动成像方法（如光栅图像相关光谱学（RICS）或数量和亮度分析（N&B））相结合。更具体地说，我们表明，可以使用 PIE-RICS 对 Venus 荧光蛋白（FP）的扩散和分子亮度进行定量测量，并将 PIE-RICS 与单点 PIE-FCS 测量进行比较。我们讨论了无串扰双光子 PIE-RICS 的优势，并通过定量比较用于双色显微镜的两种常用 FP 对 eGFP/mCherry 和 mVenus/mCherry，说明了其优势。对于 N&B 分析，我们对 PIE-FI 数据分析实施死时间校正，以便使用 PIE-NB 进行精确的分子亮度测定。然后，我们使用 PIE-NB 研究 eGFP 串联寡聚化对荧光团在细胞内成熟效率的影响。最后，我们探索了在 PIE-FI 实验中利用可用荧光寿命信息的可能性。我们对 PIE-FI 实验中的荧光寿命信息进行基于寿命的加权，从而可以使用 PIE-raster 寿命图像相关光谱学（RLICS）从 100 nM 以下定量确定分子浓度<30 pM。我们使用荧光寿命信息对串联荧光蛋白二聚体进行稳健的基于寿命的双光子 FRET 分析。最后，我们研究了使用双光子 RLICS 在细胞中从非 FRET 物种中分辨共扩散 FRET 物种的可能性。PIE-FI 增强的功能和定量结果使其成为一种广泛适用于大量有趣的生物物理研究的强大方法。
