Fluorescence imaging represents cornerstone technology for studying biological function at the cellular and molecular levels. The technology's centerpiece is a prolific collection of genetic reporters based on the green fluorescent protein (GFP) and related analogs. More than two decades of protein engineering have endowed the GFP repertoire with an incredible assortment of fluorescent proteins, allowing scientists immense latitude in choosing reporters tailored to various cellular and environmental contexts. Nevertheless, GFP and derivative reporters have specific limitations that hinder their unrestricted use for molecular imaging. These challenges have inspired the development of new reporter proteins and imaging mechanisms. Here, we review how these developments are expanding the frontiers of reporter gene techniques to enable nondestructive studies of cell function in anaerobic environments and deep inside intact animals-two important biological contexts that are fundamentally incompatible with the use of GFP-based reporters.

中文翻译：

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超越绿色荧光蛋白：厌氧和深层组织成像的生物分子报道者。

荧光成像是研究细胞和分子水平的生物学功能的基础技术。该技术的核心是大量基于绿色荧光蛋白（GFP）和相关类似物的遗传报告基因。超过二十年的蛋白质工程设计使GFP种类繁多，荧光蛋白种类繁多，使科学家在选择适合各种细胞和环境背景的报道分子时拥有极大的自由度。尽管如此，GFP和衍生物报道分子具有特定的局限性，阻碍了它们在分子成像中的无限制使用。这些挑战激发了新的报道蛋白和成像机制的发展。这里，