Piatkevich Kiryl D, Verkhusha Vladislav V
Department of Anatomy and Structural Biology, and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York, USA.
Methods Cell Biol. 2011;102:431-61. doi: 10.1016/B978-0-12-374912-3.00017-1.
Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed.
自12年前发现首个红色荧光蛋白（RFP）——DsRed以来，一系列红移荧光蛋白已被克隆，并通过生物技术开发成用于光学显微镜的单体荧光探针。还构建了几种新型单体RFP，它们要么随时间改变发射波长（称为荧光定时器），要么在短暂照射紫光后改变发射波长（称为光激活蛋白）。此外，最近还设计了具有大斯托克斯位移荧光发射的RFP。由于这些荧光探针具有专门为显微镜开发的独特激发和荧光检测条件，因此对于流式细胞术而言可能并非最佳选择。在此，我们挑选并总结了具有流式细胞术应用所需特定特性的先进橙色、红色和远红色荧光蛋白。针对商用流式细胞仪和分选仪可用的激光源计算了它们的有效亮度。确定了不同颜色荧光蛋白在多参数流式细胞术中的兼容性。还讨论了利用RFP的新型荧光共振能量转移（FRET）对、基于RFP的细胞内生物传感器及其在高通量筛选中的应用。
