Quantitative assessment of protein complexes, such as receptor clusters in the context of cellular signalling, has become a pressing objective in cell biology. The advancements in the field of single molecule fluorescence microscopy have led to different approaches for counting protein copy numbers in various cellular structures. This has resulted in an increasing interest in robust calibration protocols addressing photophysical properties of fluorescent labels and the effect of labelling efficiencies. Here, we want to give an update on recent methods for protein counting with a focus on novel calibration protocols. In this context, we discuss different types of calibration samples and identify some of the challenges arising in molecular counting experiments. Some recently published applications offer potential approaches to tackle these challenges.

蛋白质复合物的定量评估，例如细胞信号传递中的受体簇，已成为细胞生物学中的一个紧迫目标。单分子荧光显微镜领域的进步导致了计算各种细胞结构中蛋白质拷贝数的不同方法。这导致人们对解决荧光标记的光物理特性和标记效率影响的稳健校准协议越来越感兴趣。在这里，我们想对最近的蛋白质计数方法进行更新，重点是新的校准协议。在这种情况下，我们讨论了不同类型的校准样本，并确定了分子计数实验中出现的一些挑战。