Ex-dSTORM and automated quantitative image analysis of expanded filamentous structures

Area of Science:

• Biophysics
• Microscopy techniques
• Biochemistry

Background:

• Expansion microscopy (ExM) is a powerful technique to increase the effective resolution of biological samples.
• Single-molecule localization microscopy (SMLM) offers nanoscale imaging but requires specific sample preparation.
• Integrating ExM with SMLM necessitates robust protocols to maintain sample integrity and enable optimal imaging.

Purpose of the Study:

• To provide a detailed, step-by-step protocol for preparing ExM-treated biological samples for SMLM.
• To describe methods for stabilizing expanded hydrogels to prevent shrinkage during buffer exchange.
• To offer a guide for automated image analysis and expansion factor determination in ExM samples.

Main Methods:

• Sample preparation for expansion microscopy (ExM).
• Hydrogel stabilization techniques to prevent sample shrinkage.
• Application of photoswitching buffers for SMLM.
• Automated image analysis pipelines for ExM data.
• Expansion factor calculation for fiber-like structures.

Main Results:

• A reproducible protocol for preparing ExM samples for SMLM imaging.
• Successful stabilization of expanded hydrogels, preventing shrinkage.
• Demonstration of automated image analysis for efficient data processing.
• Accurate determination of expansion factors for biological structures.

Conclusions:

• The presented protocol facilitates the integration of ExM with SMLM for high-resolution imaging.
• Hydrogel stabilization is crucial for maintaining sample integrity throughout the SMLM workflow.
• Automated analysis streamlines the quantification of expansion in ExM-treated samples.