Simultaneous Interference Reflection and Total Internal Reflection Fluorescence Microscopy for Imaging Dynamic
Yazgan Tuna1, Amer Al-Hiyasat1, Jonathon Howard2
1Department of Molecular Biophysics & Biochemistry, Yale University.
Journal of Visualized Experiments : Jove
|May 23, 2022
Summary
This study introduces a novel microscopy technique combining interference reflection microscopy (IRM) with total-internal-reflection-fluorescence (TIRF) microscopy for simultaneous imaging of microtubule-associated proteins (MAPs) and microtubules. The method enables high-speed observation of molecular interactions without fluorescently labeling microtubules.
Area of Science:
- Cell Biology
- Biophysics
- Microscopy
Background:
- Direct visualization of cytoskeletal filaments and associated proteins is crucial for understanding cellular processes.
- Total-internal-reflection-fluorescence (TIRF) microscopy offers high signal-to-background but suffers from photobleaching and photodamage.
- Label-free techniques like interference reflection microscopy (IRM) avoid photobleaching but struggle with single-molecule visualization.
Purpose of the Study:
- To develop a protocol for simultaneous imaging of microtubule-associated proteins (MAPs) and dynamic microtubules in vitro.
- To overcome the limitations of existing microscopy techniques, specifically photobleaching and the need for extensive labeling.
- To enable high-speed observation of molecular interactions at the microtubule surface.
Main Methods:
- Combined interference reflection microscopy (IRM) with a commercial total-internal-reflection-fluorescence (TIRF) microscope.
- Utilized a single camera chip for simultaneous imaging of both IRM and TIRF channels, eliminating registration and synchronization issues.
- Eliminated the need for microtubule labeling and additional optical components, such as a second excitation laser.
Main Results:
- Successfully achieved simultaneous imaging of MAPs and dynamic microtubules in vitro.
- Demonstrated high-speed observation of MAPs interacting with microtubules.
- Visualized single kinesin molecules walking on dynamic microtubules using the combined IRM-TIRF setup.
Conclusions:
- The developed protocol offers a streamlined and effective method for studying microtubule dynamics and associated protein interactions.
- This combined IRM-TIRF approach overcomes photobleaching limitations and simplifies experimental setup compared to existing methods.
- The technique provides a powerful tool for visualizing single-molecule dynamics in vitro, advancing our understanding of cytoskeletal motor proteins.
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