Using Holographic Microscopy To Measure the Effect of Confinement on Crowding Agents in Lipid Vesicles

The hydrodynamic effects of macromolecular crowding inside cells are often studied in vitro by using polymers as crowding agents. 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 similar to 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 poly(ethylene 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.

Automated summary

The hydrodynamic effects of macromolecular crowding inside cells were studied using polymers as crowding agents in vitro. It was found that confinement of polymers inside cell-sized droplets affected the diffusion of small molecules. A method based on digital holographic microscopy was developed to measure the diffusion of polystyrene microspheres confined within lipid vesicles containing a high concentration of solute. The study revealed that diffusion inside and outside the vesicles was the same for sucrose or dextran prepared below the critical overlap concentration, but slower for poly(ethylene glycol) above the critical overlap concentration, suggesting potential effects of confinement on crowding agents.