Imaging Microtubules in vitro at High Resolution while Preserving their Structure

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Microtubules (MTs) are rigid cytoskeleton components but exhibit complex 3D curvature in cells.
• Mechanisms controlling MT shape in vivo remain poorly understood.
• Current imaging methods like electron microscopy (EM) and Total Internal Reflection Fluorescence (TIRF) microscopy have limitations for 3D curvature analysis.

Purpose of the Study:

• To develop a novel assay for high-resolution 3D imaging of microtubules with micrometer curvatures.
• To enable visualization of microtubules retaining their native three-dimensional shape.
• To provide a method compatible with high-resolution immunofluorescence detection.

Main Methods:

• Developed an in vitro polymerization assay using MT seeds adhered to a glass slide.
• Adapted protocols similar to TIRF microscopy for MT polymerization.
• Utilized confocal microscopy with an Airyscan module for high-resolution imaging after fixation and removal of free fluorescent molecules.

Main Results:

• Successfully visualized microtubules with micrometer-scale curvatures in their original 3D configurations.
• The developed assay preserves the native shape of microtubules.
• The method is compatible with subsequent high-resolution immunofluorescence staining.

Conclusions:

• The new assay provides a powerful tool for studying microtubule dynamics and mechanics in 3D.
• This technique overcomes limitations of existing methods for analyzing complex microtubule structures.
• Enables detailed investigation of factors influencing microtubule shape and organization within the cell.