High Precision Antibody-Free Microtubule Labeling for Expansion Microscopy

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biochemistry

Background:

• Expansion microscopy (ExM) offers nanoscale imaging but faces challenges in accurately labeling cytoskeletal filaments like microtubules.
• Existing methods can suffer from structural distortion and labeling inaccuracies during sample preparation.

Purpose of the Study:

• To develop an optimized protocol for high-precision visualization of microtubules using ExM.
• To improve the accuracy and reliability of nanoscale imaging of microtubule structures.

Main Methods:

• Combines detergent extraction to isolate microtubules and remove cellular debris.
• Utilizes NHS-ester labeling of N-termini on digested tubulin peptides, avoiding antibodies.
• Integrates into existing ExM workflows for both in vitro and cellular samples.

Main Results:

• Achieves selective removal of cytoplasmic components and membranes, preserving microtubule ultrastructure.
• Enables accurate molecular localization with minimal linkage error and high signal-to-noise ratio (SNR).
• Demonstrates effective fluorophore displacement for nanoscale resolution, dependent on expansion factor.

Conclusions:

• The optimized protocol provides a robust method for high-precision microtubule imaging in ExM.
• The labeled microtubules can serve as internal reference standards for correcting expansion factors in ExM datasets.
• This technique enhances the utility of ExM for studying cytoskeletal organization at the nanoscale.