Home › Cell Biology › Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer
Cell Biology JoVE (Open Access) Citable · DOI

Fluorescence Recovery after Merging a Droplet to Measure the Two-dimensional Diffusion of a Phospholipid Monolayer

DOI: 10.3791/53376-v
What you'll learn
  • ✓ Prepare phospholipid monolayers at flat and curved fluid interfaces
  • ✓ Measure lateral diffusion coefficients using fluorescence recovery after droplet merging
  • ✓ Quantify surface pressure effects on phospholipid mobility
Protocol

We present a new technique to measure the lateral diffusion of a surface active species at the fluid-fluid interface by merging a droplet monolayer onto a flat monolayer.

Difficulty
advanced
Total time
~4–6 hours per experiment (including monolayer preparation, imaging, and analysis)

Steps

1
Prepare phospholipid monolayer at flat air-water interface

Spread phospholipid solution onto a flat air-water interface using a Langmuir trough or equivalent apparatus. Establish the monolayer and equilibrate it at the desired surface pressure before imaging.

▶ 00:45
2
Prepare phospholipid monolayer at curved droplet surface

Form a phospholipid monolayer on the curved surface of a pendant or sessile droplet. Use equivalent lipid composition and concentration as the flat interface monolayer.

▶ 02:32
3
Image fluorescence recovery after droplet merging

Merge the droplet monolayer onto the flat monolayer while capturing fluorescence images in real time. Record recovery of fluorescence intensity in the merged region to quantify lateral diffusion.

▶ 04:00
4
Calculate diffusion coefficients from recovery data

Analyze fluorescence recovery kinetics to extract lateral diffusion coefficients for the phospholipid species. Plot diffusion as a function of surface pressure.

▶ 05:17
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