Adenosine triphosphate (ATP) is the central energy carrier of all cells and knowledge on the dynamics of the concentration of ATP ([ATP]) provides important insights into the energetic state of a cell. Several genetically encoded fluorescent nanosensors for ATP were developed, which allow following the cytosolic [ATP] at high spatial and temporal resolution using fluorescence microscopy. However, to calibrate the fluorescent signal to [ATP] has remained challenging. To estimate basal cytosolic [ATP] ([ATP]₀) in astrocytes, we here took advantage of two ATP nanosensors of the ATeam-family (ATeam1.03; ATeam1.03YEMK) with different affinities for ATP. Altering [ATP] by external stimuli resulted in characteristic pairs of signal changes of both nanosensors, which depend on [ATP]₀. Using this dual nanosensor strategy and epifluorescence microscopy, [ATP]₀ was estimated to be around 1.5 mM in primary cultures of cortical astrocytes from mice. Furthermore, in astrocytes in acutely isolated cortical slices from mice expressing both nanosensors after stereotactic injection of AAV-vectors, 2-photon microscopy revealed [ATP]₀ of 0.7 mM to 1.3 mM. Finally, the change in [ATP] induced in the cytosol of cultured cortical astrocytes by application of azide, glutamate, and an increased extracellular concentration of K⁺ were calculated as −0.50 mM, −0.16 mM, and 0.07 mM, respectively. In summary, the dual nanosensor approach adds another option for determining the concentration of [ATP] to the increasing toolbox of fluorescent nanosensors for metabolites. This approach can also be applied to other metabolites when two sensors with different binding properties are available.

三磷酸腺苷 (ATP) 是所有细胞的中心能量载体，有关 ATP ([ATP]) 浓度动态的知识为了解细胞的能量状态提供了重要的见解。开发了几种针对 ATP 的基因编码荧光纳米传感器，允许使用荧光显微镜以高空间和时间分辨率跟踪胞质 [ATP]。然而，校准 [ATP] 的荧光信号仍然具有挑战性。为了估计星形胶质细胞中的基础胞质 [ATP] ([ATP] ₀ )，我们利用了 ATeam 系列的两个 ATP 纳米传感器（ATeam1.03；ATeam1.03YEMK），它们对 ATP 具有不同的亲和力。通过外部刺激改变 [ATP] 导致两个纳米传感器的信号变化的特征对，这取决于 [ATP] ₀ 。使用这种双纳米传感器策略和落射荧光显微镜，在小鼠皮质星形胶质细胞的原代培养物中，[ATP] ₀估计约为 1.5 mM。此外，在立体定向注射 AAV 载体后，在表达两种纳米传感器的小鼠中急性分离的皮质切片中的星形胶质细胞中，双光子显微镜显示 [ATP] ₀为 0.7 mM 至 1.3 mM。最后，通过应用叠氮化物、谷氨酸和增加细胞外浓度的 K ⁺在培养的皮质星形胶质细胞的细胞质中诱导的 [ATP] 变化分别计算为 -0.50 mM、-0.16 mM 和 0.07 mM。总之，双纳米传感器方法为不断增加的代谢物荧光纳米传感器工具箱添加了另一种测定 [ATP] 浓度的选项。当具有不同结合特性的两个传感器可用时，这种方法也可以应用于其他代谢物。