Going live: A comparative analysis of the suitability of the RFP derivatives RedStar, mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites
Going live: A comparative analysis of the suitability of the RFP derivatives RedStar, mCherry and tdTomato for intravital and in vitro live imaging of Plasmodium parasites
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DOI：
10.1002/biot.200900035
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发表时间：
2009-06-01
影响因子：
4.7
通讯作者：
Heussler, Volker T.
中科院分区：
文献类型：
--
作者：
Graewe, Stefanie;Retzlaff, Silke;Heussler, Volker T.
荧光蛋白已被证明是重要的工具，在体外活的寄生虫成像和成像的寄生虫内的活体显微镜。我们观察到，红色荧光转基因疟疾寄生虫的啮齿动物，伯氏疟原虫红星，是适合在体外活成像实验，但漂白后，在活体成像实验中使用小鼠的照明迅速。因此，我们已经产生了两个额外的转基因寄生虫线表达的新的红色荧光蛋白tdTomato和mCherry，这已被报道是更多的光稳定性比第一和第二基因定量红色荧光蛋白，包括红星。我们使用共聚焦显微镜和宽视野显微镜，比较了所有三种红色荧光寄生虫系在伯氏疟原虫血液和肝脏寄生虫阶段的体外活体和活体成像中的用途。虽然tdTomato几乎与RedStar一样快速漂白，但mCherry显示出改善的光稳定性，并且在所有实验中都是明亮的。
Fluorescent proteins have proven to be important tools for in vitro live imaging of parasites and for imaging of parasites within the living host by intravital microscopy. We observed that a red fluorescent transgenic malaria parasite of rodents, Plasmodium berghei-RedStar, is suitable for in vitro live imaging experiments but bleaches rapidly upon illumination in intravital imaging experiments using mice. We have therefore generated two additional transgenic parasite lines expressing the novel red fluorescent proteins tdTomato and mCherry, which have been reported to be much more photostable than first- and second-gene ration red fluorescent proteins including RedStar. We have compared all three red fluorescent parasite lines for their use in in vitro live and intravital imaging of P. berghei blood and liver parasite stages, using both confocal and wide-field microscopy. While tdTomato bleached almost as rapidly as RedStar, mCherry showed improved photostability and was bright in all experiments performed.