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Botany JoVE (Open Access) Citable · DOI

A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

DOI: 10.3791/55331-v
What you'll learn
  • ✓ Assemble a simple chamber for long-term confocal imaging of plant seedlings
  • ✓ Perform high-resolution time-lapse imaging of root and hypocotyl development over 3 days
  • ✓ Use perfluorodecalin as an immersion medium for extended live-cell imaging
  • ✓ Compare growth dynamics in imaging chambers versus traditional agar plate culture
Protocol

Presented here is a simple technique for high-resolution confocal time-lapse imaging of root and hypocotyl development for up to 3 days using high numerical-aperture objectives and perfluorodecalin as an immersion medium.

Difficulty
intermediate
Total time
~3 days (continuous imaging); ~1-2 hrs chamber preparation and setup per experiment
Model organism
Arabidopsis thaliana

Steps

1
Prepare seedling growth imaging chambers

Construct simple imaging chambers using standard materials to house seedlings. Assemble components to enable long-term confocal microscopy with minimal environmental disruption.

▶ 00:50
2
Mount seedlings and apply immersion medium

Position seedlings in prepared chambers and apply perfluorodecalin as an immersion medium to enable high-resolution imaging with high numerical-aperture objectives over extended periods.

▶ 04:01
3
Acquire time-lapse confocal image sequences

Perform continuous or time-interval confocal microscopy to capture root and hypocotyl development over multiple days, maintaining optical clarity and specimen viability.

▶ 05:40
4
Compare growth in chambers versus agar plates

Analyze and compare root growth rates and developmental dynamics between seedlings imaged in chambers and those cultured on traditional agar plates to validate the imaging approach.

▶ 07:15
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