The physical and chemical environment inside cells is of fundamental importance to all life but has traditionally been difficult to determine on a subcellular basis. Here we combine cutting-edge genomically integrated FRET biosensing to readout localized molecular crowding in single live yeast cells. Confocal microscopy allows us to build subcellular crowding heatmaps using ratiometric FRET, while whole-cell analysis demonstrates crowding is reduced when yeast is grown in elevated glucose concentrations. Simulations indicate that the cell membrane is largely inaccessible to these sensors and that cytosolic crowding is broadly uniform across each cell over a timescale of seconds. Millisecond single-molecule optical microscopy was used to track molecules and obtain brightness estimates that enabled calculation of crowding sensor copy numbers. The quantification of diffusing molecule trajectories paves the way for correlating subcellular processes and the physicochemical environment of cells under stress.

中文翻译：

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单个真核细胞中的分子拥挤：使用细胞环境生物传感和单分子光学显微镜探查对细胞外离子强度，局部葡萄糖条件和传感器拷贝数的依赖性

细胞内部的物理和化学环境对整个生命至关重要，但传统上很难以亚细胞为基础进行确定。在这里，我们结合了最先进的基因组整合的FRET生物传感技术，以读出单个活酵母细胞中的局部分子拥挤情况。共聚焦显微镜使我们能够使用比率式FRET建立亚细胞拥挤热图，而全细胞分析表明，当酵母在葡萄糖浓度升高的情况下生长时，拥挤会减少。模拟表明，这些传感器在很大程度上无法进入细胞膜，并且在数秒的时间内，每个细胞的胞浆拥挤情况大致相同。毫秒单分子光学显微镜用于跟踪分子并获得亮度估计值，从而可以计算拥挤的传感器拷贝数。