Home › Cell Biology › Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules
Cell Biology JoVE (Open Access) Citable · DOI

Implementation of Interference Reflection Microscopy for Label-free, High-speed Imaging of Microtubules

DOI: 10.3791/59520-v
What you'll learn
  • ✓ Set up interference reflection microscopy on a standard fluorescence microscope
  • ✓ Acquire label-free, high-contrast images of microtubule dynamics in vitro
  • ✓ Align optical path and optimize aperture diaphragm for maximum contrast
  • ✓ Process IRM images to enhance microtubule visualization and dynamics
Protocol

Biopharma Insights This protocol is a guide for implementing interference reflection microscopy on a standard fluorescence microscope for label-free, high-contrast, high-speed imaging of microtubules using in vitro surfaces assays.

Difficulty
advanced
Total time
~4–6 hours (including microscope alignment, chamber preparation, and imaging acquisition)

Steps

1
Modify microscope and prepare imaging chamber

Install interference reflection optics on the fluorescence microscope and assemble the sample chamber with appropriate surfaces for in vitro microtubule assays.

▶ 00:46
2
Align optical path for interference reflection

Optimize alignment of the illumination and detection paths to achieve proper constructive/destructive interference conditions for high-contrast microtubule imaging.

▶ 02:32
3
Image stabilized microtubules as control

Acquire baseline images of immobilized microtubules to verify system performance and confirm proper interference reflection contrast.

▶ 03:43
4
Image dynamic microtubule polymerization

Capture high-speed video of actively polymerizing and depolymerizing microtubules to demonstrate real-time dynamics without fluorescent labels.

▶ 04:43
5
Optimize aperture diaphragm for contrast

Systematically adjust aperture diaphragm size to maximize signal-to-noise ratio and image contrast during microtubule observation.

▶ 06:28
6
Process images to enhance microtubule visualization

Apply image processing algorithms to increase contrast and clarify microtubule dynamics in raw IRM data for quantitative analysis.

▶ 08:11
💬 Comments coming soon