Department of Bioengineering, University of California, Berkeley, CA 94720, USA.
Bioorg Med Chem. 2011 Feb 1;19(3):1030-40. doi: 10.1016/j.bmc.2010.07.015. Epub 2010 Jul 30.
A major challenge in cell biology is to elucidate molecular mechanisms that underlie the spatio-temporal control of cellular processes. These studies require microscope imaging techniques and associated optical probes that provide high-contrast and high-resolution images of specific proteins and their complexes. Auto-fluorescence however, can severely compromise image contrast and represents a fundamental limitation for imaging proteins within living cells. We have previously shown that optical switch probes and optical lock-in detection (OLID) image microscopy improve image contrast in high background environments. Here, we present the design, synthesis, and characterization of amino-reactive and cell permeable optical switches that integrate the highly fluorescent fluorophore, tetramethylrhodamine (TMR) and spironaphthoxazine (NISO), a highly efficient optical switch. The NISO moiety in TMR-NISO undergoes rapid and reversible, excited-state driven transitions between a colorless spiro (SP)-state and a colored merocyanine (MC)-state in response to irradiation with 365 and >530nm light. In the MC-state, the TMR (donor) emission is almost completely extinguished by Förster resonance energy transfer (FRET) to the MC probe (acceptor), whereas in the colorless SP-state, the quantum yield for TMR fluorescence is maximal. Irradiation of TMR-NISO with a defined sequence of 365 and 546nm manipulates the levels of SP and MC with concomitant modulation of FRET efficiency and the TMR fluorescence signal. High fidelity optical switching of TMR fluorescence is shown for TMR-NISO probes in vitro and for membrane permeable TMR-NISO within living cells.
细胞生物学的一个主要挑战是阐明细胞过程时空控制的分子机制。这些研究需要显微镜成像技术和相关的光学探针，以提供特定蛋白质及其复合物的高对比度和高分辨率图像。然而，自发荧光会严重降低图像对比度，是对活细胞内蛋白质进行成像的一个基本限制。我们之前已经表明，光学开关探针和光学锁相检测（OLID）显微镜可以提高高背景环境下的图像对比度。在这里，我们设计、合成和表征了具有反应性的和细胞渗透性的光学开关，这些光学开关整合了高度荧光的荧光团四甲基罗丹明（TMR）和螺萘并恶嗪（NISO），这是一种高效的光学开关。TMR-NISO 中的 NISO 部分在受到 365nm 和>530nm 光照射时，在无颜色的螺（SP）态和有色的甲川（MC）态之间快速且可逆地进行激发态驱动的转变。在 MC 态中，TMR（供体）的发射几乎完全被Förster 共振能量转移（FRET）到 MC 探针（受体）而熄灭，而在无色的 SP 态中，TMR 荧光的量子产率最大。用 365nm 和 546nm 的特定序列照射 TMR-NISO，可以操纵 SP 和 MC 的水平，同时调制 FRET 效率和 TMR 荧光信号。在体外 TMR-NISO 探针和活细胞内可渗透的 TMR-NISO 中都显示出 TMR 荧光的高保真光学开关。
