Microtubules as a versatile reference standard for expansion microscopy

Area of Science:

• Biotechnology
• Microscopy
• Cell Biology

Background:

• Expansion microscopy (ExM) is advancing, requiring standardized validation methods.
• Current ExM validation structures lack consensus, hindering technique development.

Purpose of the Study:

• To propose and validate microtubules as a universal standard for ExM validation.
• To establish protocols for using microtubules across different ExM resolution scales.

Main Methods:

• Immunostaining of cellular microtubules for validation of techniques with <50 nm resolution.
• In vitro assembly and imaging of microtubules for validation of techniques with ~10 nm resolution.
• Labeling tubulin peptide chains and analyzing single tubulin molecules for nanometer-scale techniques.

Main Results:

• Microtubule diameter is suitable for validating ExM resolutions.
• Protocols for in vitro microtubule assembly and cellular extraction are presented.
• Multiple methods demonstrate microtubule utility for ExM validation.

Conclusions:

• Microtubules offer a versatile and reliable structure for ExM validation.
• The proposed methods support the standardization of ExM technology assessment.
• Microtubules are valuable for validating ExM and related super-resolution imaging technologies.