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Related Experiment Video

Updated: Jul 12, 2025

Label-Retention Expansion Microscopy LR-ExM Enables Super-Resolution Imaging and High-Efficiency Labeling

Label-Retention Expansion Microscopy LR-ExM Enables Super-Resolution Imaging and High-Efficiency Labeling

Published on: October 11, 2022

Immunolabel-First-Expand-Later Expansion Microscopy Approach Using Stable STED Dyes.

Dong Kong¹, Delgermaa Luvsanjav¹, Jadranka Loncarek²

¹Cancer Innovation Laboratory, NIH/NCI/CCR, Frederick, MD, USA.

Methods in Molecular Biology (Clifton, N.J.)

|October 19, 2023

Summary

This study introduces a novel expansion microscopy method using stable STED dyes for robust protein detection in centrioles and cilia. This technique improves antibody binding after sample expansion, enabling nanoscale imaging of key proteins.

Keywords:

Abberior STAR ORANGE Abberior STAR RED Centriole Centrosome Cep164 Cilia Expansion STED

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Area of Science:

Cell Biology
Microscopy Techniques
Molecular Imaging

Background:

Expansion microscopy (ExM) enables nanoscale imaging of cellular structures like centrioles, centrosomes, and cilia.
Current ExM protocols often face challenges with antibody binding to proteins after sample expansion and denaturation.
Precise localization of centrosomal and ciliary proteins is crucial for understanding cell function.

Purpose of the Study:

To develop and validate an expansion microscopy protocol using stable STED dyes for improved protein detection.
To overcome limitations in primary antibody binding to proteins post-denaturation in ExM.
To enable robust nanoscale imaging of centriolar and ciliary proteins using an 'immunolabel-first-expand-later' approach.

Main Methods:

Utilized two stable STED dyes compatible with expansion microscopy protocols.
Implemented an 'immunolabel-first-expand-later' strategy, performing immunolabeling before sample expansion.
Validated dye stability through crosslinking, polymerization, and denaturation steps of the ExM protocol.

Main Results:

Demonstrated the stability of STED dyes throughout the expansion microscopy workflow.
Achieved robust detection of immunolabeled proteins using both conventional and STED microscopy after expansion.
Successfully analyzed the localization of the centriole appendage protein Cep164 and the ciliary protein ARL13B.

Conclusions:

The developed ExM protocol with stable STED dyes offers a reliable method for nanoscale protein localization.
This approach effectively addresses the challenge of antibody accessibility after protein denaturation in ExM.
The method provides a valuable tool for advancing the study of centrioles, centrosomes, and cilia.