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Cell division, aging, and stress recovery triggers spatial reorganization ofcellular components in the cytoplasm, including membrane bound organelles, withmolecular changes in their compositions and structures. However, it is notclear how these events are coordinated and how they integrate with regulationof molecular crowding. We use the budding yeast Saccharomyces cerevisiae as amodel system to study these questions using recent progress in opticalfluorescence microscopy and crowding sensing probe technology. We used aF\"{o}rster Resonance Energy Transfer (FRET) based sensor, illuminated byconfocal microscopy for high throughput analyses and Slimfield microscopy forsingle-molecule resolution, to quantify molecular crowding. We determinecrowding in response to cellular growth of both mother and daughter cells, inaddition to osmotic stress, and reveal hot spots of crowding across the budneck in the burgeoning daughter cell. This crowding might be rationalized bythe packing of inherited material, like the vacuole, from mother cells. Wediscuss recent advances in understanding the role of crowding in cellularregulation and key current challenges and conclude by presenting our recentadvances in optimizing FRET-based measurements of crowding whilstsimultaneously imaging a third color, which can be used as a marker that labelsorganelle membranes. Our approaches can be combined with synchronised cellpopulations to increase experimental throughput and correlate molecularcrowding information with different stages in the cell cycle.

Investigating molecular crowding during cell division in budding yeast with FRET