Abstract In eukaryotes, intracellular physicochemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion, and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalised in cells is tracked and physicochemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Förster Resonance Energy Transfer (FRET) biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multidimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterise in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae . It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalise fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells, and discuss limitations of this approach including impairment of cellular function.

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo