Centre de Biochimie Structurale, INSERM U554, CNRS UMR 5048, Université Montpellier 1 and 2, F-34090 Montpellier, France.
Anal Biochem. 2011 Dec 15;419(2):250-9. doi: 10.1016/j.ab.2011.08.017. Epub 2011 Aug 22.
Quantification of promoter activity or protein expression in gene regulatory networks is generally achieved via measurement of fluorescent protein (FP) intensity, which is related to the true FP concentration by an unknown scaling factor, thereby limiting analysis and interpretation. Here, using approaches originally developed for eukaryotic cells, we show that two-photon (2p) fluorescence fluctuation microscopy, specifically scanning number and brightness (sN&B) analysis, can be applied to determine the absolute concentrations of diffusing FPs in live bacterial cells. First, we demonstrate the validity of the approach, despite the small size of the bacteria, using the central pixels and spatial averaging. We established the lower detection limit at or below 75 nM (~3 molecules of FP/vol(ex)) and the upper detection limit at approximately 10 μM, which can be extended using intensity measurements. We found that the uncertainty inherent in our measurements (<5%) was smaller than the high cell-cell variations observed for stochastic leakage from FP fusions of the lac promoter in the repressed state or the 10 to 25% variation observed on induction. This demonstrates that a reliable and absolute measure of transcriptional noise can be made using our approach, which should make it particularly appropriate for the investigation of stochasticity in gene expression networks.
在基因调控网络中，通常通过测量荧光蛋白（FP）的强度来量化启动子活性或蛋白质表达，而该强度与真实的 FP 浓度通过一个未知的缩放因子相关联，从而限制了分析和解释。在这里，我们使用最初为真核细胞开发的方法，表明双光子（2p）荧光波动显微镜，特别是扫描数量和亮度（sN&B）分析，可以应用于确定活细菌细胞中扩散 FP 的绝对浓度。首先，我们通过中心像素和空间平均来证明该方法的有效性，尽管细菌的体积很小。我们确定了下限检测值在 75 nM 或以下（~3 个 FP 分子/细胞体积），上限检测值在约 10 μM，可通过强度测量进行扩展。我们发现，我们的测量值中固有的不确定性（<5%）小于 lac 启动子在抑制状态下从 FP 融合随机泄漏或诱导时观察到的 10%至 25%的高细胞间变化。这表明，使用我们的方法可以对转录噪声进行可靠和绝对的测量，这使其特别适合于基因表达网络中的随机性研究。
