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The physical and chemical environment inside cells is of fundamentalimportance to all life but has traditionally been difficult to determine on asubcellular basis. Here we combine cutting-edge genomically integrated FRETbiosensing to readout localized molecular crowding in single live yeast cells.Confocal microscopy allows us to build subcellular crowding heatmaps usingratiometric FRET, while whole-cell analysis demonstrates crowding is reducedwhen yeast is grown in elevated glucose concentrations. Simulations indicatethat the cell membrane is largely inaccessible to these sensors and thatcytosolic crowding is broadly uniform across each cell over a timescale ofseconds. Millisecond single-molecule optical microscopy was used to trackmolecules and obtain brightness estimates that enabled calculation of crowdingsensor copy numbers. The quantification of diffusing molecule trajectoriespaves the way for correlating subcellular processes and the physicochemicalenvironment of cells under stress.

Molecular crowding in single eukaryotic cells: using cell environment  biosensing and single-molecule optical microscopy to probe dependence on  extracellular ionic strength, local glucose conditions, and sensor copy  number