Multicamera simultaneous total internal reflection and interference reflection microscopy.

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Interference Reflection Microscopy (IRM) images biological structures by analyzing light interference.
• IRM has been used to visualize microtubules, essential biofilaments.
• Simultaneous imaging of microtubules and associated proteins is often desired but challenging.

Purpose of the Study:

• To develop a simple and cost-effective method for simultaneous Interference Reflection Microscopy (IRM) and single-molecule total internal reflection fluorescence (TIRF) imaging.
• To enable the simultaneous visualization of microtubules and microtubule-interacting proteins.

Main Methods:

• A standard multicolour total internal reflection fluorescence (TIRF) microscope was modified.
• The modification allows for simultaneous high-speed IRM and single-molecule TIRF imaging.
• Separate cameras were used for IRM and TIRF channels for independent parameter optimization.

Main Results:

• The modified microscope successfully enabled simultaneous high-speed IRM and single-molecule TIRF imaging.
• Unlabelled microtubules and GFP-labelled end-binding protein EB1 were imaged concurrently.
• EB1 protein 'comets' at the tips of polymerizing microtubules were visualized.

Conclusions:

• The described modification provides an accessible and affordable way to achieve simultaneous IRM and TIRF imaging.
• This technique enhances the ability to study microtubule dynamics and protein interactions in real-time.
• The method is easily implementable in laboratories with existing fluorescence microscopes.