  The hydrodynamic effects of macromolecular crowding inside cells is often
studied in vitro by using polymers as crowding reagents. Confinement of
polymers inside cell-sized droplets has been shown to affect the diffusion of
small molecules. Here we develop a method, based on digital holographic
microscopy, to measure the diffusion of polystyrene microspheres that are
confined within lipid vesicles containing a high concentration of solute. We
apply the method to three solutes of varying complexity: sucrose, dextran, and
PEG, prepared at ~7 % (w/w). We find that diffusion inside and outside the
vesicles is the same when the solute is sucrose or dextran that is prepared
below the critical overlap concentration. For polyethylene glycol, which is
present at a concentration higher than the critical overlap concentration, the
diffusion of microspheres inside vesicles is slower, hinting at the potential
effects of confinement on crowding agents.
