Genetically-encoded biosensors based on a single fluorescent protein are widely used to visualize analyte levels or enzymatic activities in cells, though usually to monitor relative changes rather than absolute values. We report photochromism-enabled absolute quantification (PEAQ) biosensing, a method that leverages the photochromic properties of biosensors to provide an absolute measure of the analyte concentration or activity. We develop proof-of-concept photochromic variants of the popular GCaMP family of Ca²⁺ biosensors, and show that these can be used to resolve dynamic changes in the absolute Ca²⁺ concentration in live cells. We also show how our method can be expanded to fast imaging with reduced illumination intensities or to situations where the absolute illumination intensities are unknown. In principle, PEAQ biosensing can be applied to other biosensors with photochromic properties, thereby expanding the possibilities for fully quantitative measurements in complex and dynamic systems.

中文翻译：

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使用光致变色单荧光团生物传感器绝对测量细胞活动

基于单一荧光蛋白的基因编码生物传感器被广泛用于可视化细胞中的分析物水平或酶活性，但通常用于监测相对变化而不是绝对值。我们报告了启用光致变色的绝对定量 (PEAQ) 生物传感，这是一种利用生物传感器的光致变色特性来提供分析物浓度或活性的绝对测量的方法。我们开发了流行的 GCaMP 系列 Ca ²⁺生物传感器的概念验证光致变色变体，并表明这些变体可用于解决绝对 Ca ^{2+ 的}动态变化活细胞中的浓度。我们还展示了如何将我们的方法扩展到照明强度降低的快速成像或绝对照明强度未知的情况。原则上，PEAQ 生物传感可以应用于其他具有光致变色特性的生物传感器，从而扩展在复杂和动态系统中进行完全定量测量的可能性。