# Dual-Color Expansion Microscopy of Membrane Proteins Using Bioorthogonal Labeling

## TL;DR

This paper introduces a new method combining bioorthogonal labeling and expansion microscopy to achieve high-resolution imaging of membrane proteins at the nanoscale.

## Contribution

The novel approach integrates noncanonical amino acid labeling with expansion microscopy for dual-color super-resolution imaging.

## Key findings

- The method enables precise visualization of Na,K-ATPase α1 and β1 subunits in expanded HEK 293T cells.
- Validation via STED imaging confirms the accuracy of ncAA labeling in unexpanded cells.
- The framework supports multiplexed, nanoscale biological imaging.

## Abstract

With recent advances in fluorescence microscopy, resolution
is
often limited by the size of the label and the resulting linkage error,
rather than the microscope itself. Site-specific incorporation of
noncanonical amino acids (ncAAs) combined with bioorthogonal click
chemistry provides a powerful tool for fluorescent protein labeling,
overcoming the spatial uncertainty inherent to antibody-based probes.
Here, we present a method to further improve labeling precision by
combining ncAA labeling with expansion microscopy (ExM) for dual-color
super-resolution imaging. After optimizing labeling procedures and
fluorophore selection, we visualize and resolve the nanoscale distribution
of Na,K-ATPase α1 and β1 subunits
in expanded HEK 293T cells. We validate our approach by super-resolution
STED imaging of the ncAA labeled β1 subunit in unexpanded
cells. This work presents a strong framework for multiplexed, high-resolution
imaging, suggesting that ncAA labeling combined with ExM enables biological
imaging at the nanometer scale.