Home › Microbiology › Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking
Microbiology JoVE (Open Access) Citable · DOI

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

DOI: 10.3791/51177-v
What you'll learn
  • ✓ Prepare live bacterial cells on slides for PALM microscopy imaging
  • ✓ Configure microscopy hardware and acquire photoactivated localization movies
  • ✓ Analyze PALM data to track single protein-DNA binding events quantitatively
  • ✓ Interpret temporal dynamics of protein-DNA interactions in living cells
Protocol

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Difficulty
advanced
Total time
~4–6 hours per experiment (sample preparation ~1 hr, microscopy acquisition ~2–3 hrs, analysis ~1–2 hrs)
Model organism
Escherichia coli (bacterium)
Biosafety
BSL-1

Steps

1
Prepare bacterial cells on microscopy slide

Mount live E. coli cells expressing photoactivatable fluorescent protein-DNA binding partners on a microscope slide with appropriate mounting medium to maintain cell viability during imaging.

▶ 01:32
2
Configure microscope hardware and camera settings

Set up photoactivation laser, detection optics, and camera parameters (gain, exposure, ROI) to optimize single-molecule detection sensitivity and minimize background noise.

▶ 04:00
3
Acquire photoactivated localization microscopy movies

Record time-lapse PALM image sequences by alternating photoactivation pulses with high-speed widefield fluorescence detection to capture single fluorophore blinking events in live cells.

▶ 06:43
4
Analyze PALM movies to quantify protein trajectories

Process raw PALM image stacks to localize individual photons, link detections across frames, and reconstruct single-molecule trajectories, yielding binding dwell times and diffusion coefficients.

▶ 08:16
5
Interpret representative results from single-cell data

Examine tracked Pol1-DNA binding trajectories in individual E. coli cells and extract kinetic parameters to assess protein-DNA interaction specificity and dynamics in the living bacterial nucleus.

▶ 12:48
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