Studies have reported on the ability of green fluorescent proteins to photoconvert into a red fluorescent form under various conditions, such as the presence of oxidants, hypoxia, as well as under benign conditions using irradiation with a 405 nm laser. Here, we show that in Saccharomyces cerevisiae yeast green fluorescent protein (GFP) (S65T) fused to different cellular proteins can easily photoconvert into a red form when cells are grown in media with nonfermentable carbon sources. This photoconversion occurs during standard microscopy between glass slide and coverslip but is completely prevented by imaging on pads of solid medium or in a large volume of medium on an inverted microscope. The observed effect was due to rapid hypoxia of cells with respiratory metabolism in standard conditions for upright microscopy. Photoconversion could be prevented by antioxidant treatment, suggesting that it proceeds via the effects of reactive oxidative species emerging in response to oxygen deficiency. Our results show the need for caution during upright microscopy imaging in conditions where there is active respiration and demonstrate simple approaches to prevent unwanted GFP photoconversion. They also provide easy means of performing photoconversion experiments on existing GFP‐bearing cell lines, at least in the case of yeast.

中文翻译：

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通过碳源和氧气可用性在荧光显微镜下调节 GFP 的绿色到红色光转换

研究报告了绿色荧光蛋白在各种条件下（例如存在氧化剂、缺氧）以及在使用 405 nm 激光照射的良性条件下光转换为红色荧光形式的能力。在这里，我们表明在酿酒酵母中当细胞在具有不可发酵碳源的培养基中生长时，酵母绿色荧光蛋白 (GFP) (S65T) 与不同的细胞蛋白融合后可以很容易地光转化为红色形式。这种光转换发生在玻璃载玻片和盖玻片之间的标准显微镜检查过程中，但通过在固体培养基垫或倒置显微镜上的大量培养基上成像可以完全阻止。观察到的效果是由于在直立显微镜的标准条件下具有呼吸代谢的细胞的快速缺氧。抗氧化处理可以防止光转化，这表明它是通过响应缺氧而出现的活性氧化物质的作用进行的。我们的结果表明，在呼吸活跃的情况下，在直立显微镜成像过程中需要谨慎，并展示了防止不需要的 GFP 光转换的简单方法。它们还提供了在现有的带有 GFP 的细胞系上进行光转换实验的简单方法，至少在酵母的情况下是这样。