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Microscopy & Imaging JoVE (Open Access) Citable · DOI

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

DOI: 10.3791/53158-v
What you'll learn
  • ✓ Prepare protein-functionalized planar supported lipid bilayers on glass substrates
  • ✓ Measure protein mobility and density using single-molecule fluorescence microscopy
  • ✓ Build and optimize a Total Internal Reflection (TIRF) microscope system
  • ✓ Quantify bilayer fluidity and characterize fluorescent protein behavior
Protocol

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

Difficulty
advanced
Total time
~3–4 hours per sample (bilayer preparation through imaging acquisition)

Steps

1
Understand single-molecule bilayer imaging principles

Review conceptual foundations of protein-functionalized planar supported lipid bilayers and their application to studying protein dynamics at the single-molecule level.

▶ 00:23
2
Prepare lipid vesicles from stock solutions

Assemble lipid vesicles as precursor material for bilayer deposition, establishing the starting material composition and size.

▶ 01:36
3
Form supported bilayer on glass substrate

Deposit and rupture vesicles onto glass surface to create a continuous, protein-functionalized planar bilayer suitable for microscopy.

▶ 04:22
4
Apply Total Internal Reflection Microscopy principles

Understand the optical basis of TIRF illumination for selective excitation of fluorophores near the bilayer surface with minimal background.

▶ 08:12
5
Construct noise-optimized TIRF microscope system

Assemble optical and mechanical components of the Total Internal Reflection microscope to achieve high spatiotemporal resolution imaging.

▶ 10:44
6
Measure laser power and optimize illumination

Calibrate incident laser power and verify proper TIRF alignment to ensure consistent, artifact-free single-molecule detection.

▶ 12:41
7
Quantify bilayer fluidity from protein trajectories

Track individual protein lateral diffusion within the bilayer and extract mobility metrics from single-molecule trajectories.

▶ 15:38
8
Determine protein density on bilayer surface

Analyze spatiotemporal distribution and counting statistics of fluorescent proteins to establish surface density quantitatively.

▶ 16:39
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