Home › Cell Biology › Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules
Cell Biology JoVE (Open Access) Citable · DOI

Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

DOI: 10.3791/1542-v
What you'll learn
  • ✓ Assemble and activate a microfluidic cell for biomolecule immobilization
  • ✓ Configure automated confocal microscopy for single-molecule FRET measurements
  • ✓ Acquire and interpret time-resolved fluorescence traces from surface-immobilized DNA
Protocol

Biopharma Insights In this article we describe how we obtain FRET traces from individual DNA molecules immobilized to a surface using an automated scanning confocal microscope.

Difficulty
advanced
Total time
~4–6 hours per experiment (including surface preparation, buffer equilibration, and scanning)
Biosafety
BSL-1

Steps

1
Assemble the microfluidic cell

Construct the microfluidic chamber by integrating coverslips, spacers, and inlet/outlet connectors. Ensure proper sealing to prevent leakage during subsequent immobilization and imaging steps.

▶ 01:44
2
Activate surface for molecule immobilization

Chemically treat the microfluidic cell surface using appropriate surface activation protocols (e.g., silanization or biotin–streptavidin coating) to enable stable attachment of biomolecules.

▶ 05:35
3
Prepare imaging buffer and load sample

Formulate the imaging buffer with appropriate pH, osmolarity, and oxygen-scavenging components. Introduce the buffer and sample DNA into the microfluidic cell.

▶ 07:31
4
Record time-resolved FRET traces

Initiate automated confocal scanning to acquire single-molecule fluorescence intensity time series from individual immobilized DNA molecules, generating FRET efficiency traces.

▶ 10:03
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