Cayman Chemical Company, Ann Arbor, MI, United States.
Michigan State University, East Lansing, MI, United States.
Methods Enzymol. 2020;639:389-411. doi: 10.1016/bs.mie.2020.04.058.
Fluorescence cell imaging provides a powerful tool to study biological processes including regulation, protein-protein interaction, trafficking, development, cellular structure and morphology, to name a few. Complimentary to fluorescent proteins (FPs), the development of multiple site-selective labeling techniques offer choice and flexibility in selection of fluorophores for optimal experimental design. Near-infrared (NIR) labels are highly desired since they enable deeper imaging depths and cleaner optical windows. Photochromic labels are also desirable since they provide the capability to control the fluorescence "turn-ON" and in some cases "turn-OFF" functionality. In addition, no-wash labeling techniques can greatly simplify experimental procedures and offer real-time imaging options. Also, compared to most of the regular FPs, these systems are often matured rapidly and do not need molecular oxygen for activation. Here, we present a no-wash photochromic NIR fluorescence live cell imaging approach. This method uses engineered human Cellular Retinol Binding Protein II (hCRBPII) as a genetically encodable tag and a solvatochromic dye FR-1V as the fluorophore. At the heart of this system, a photo-triggered switching between NIR "OFF" and "ON" modes provide spatiotemporal control for subcellular fluorescence imaging.
荧光细胞成像提供了一种强大的工具，可用于研究包括调控、蛋白质-蛋白质相互作用、运输、发育、细胞结构和形态等在内的生物学过程。除了荧光蛋白 (FPs)，多种位点选择性标记技术的发展为最佳实验设计中荧光团的选择提供了更多的选择和灵活性。近红外 (NIR) 标记物是非常需要的，因为它们能够实现更深的成像深度和更清洁的光学窗口。光致变色标记物也是需要的，因为它们提供了控制荧光“开启”和在某些情况下“关闭”功能的能力。此外，无需洗涤的标记技术可以大大简化实验程序，并提供实时成像选项。与大多数常规的 FPs 相比，这些系统通常能够快速成熟，并且不需要分子氧来激活。在这里，我们提出了一种无需洗涤的光致变色近红外荧光活细胞成像方法。该方法使用工程化的人细胞视黄醇结合蛋白 II (hCRBPII) 作为可遗传编码的标签和溶剂变色染料 FR-1V 作为荧光团。在这个系统的核心，光触发的 NIR“关闭”和“开启”模式之间的切换为亚细胞荧光成像提供了时空控制。
