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Updated: Jan 21, 2026
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Label-Retention Expansion Microscopy LR-ExM Enables Super-Resolution Imaging and High-Efficiency Labeling
Published on: October 11, 2022
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Bioorthogonal labeling with tetrazine-dyes for super-resolution microscopy
Gerti Beliu1, Andreas J Kurz1, Alexander C Kuhlemann1
11Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Am Hubland, 97074 Würzburg, Germany.
Communications Biology
|July 26, 2019
Summary
Genetic code expansion technology enables precise protein labeling using functionalized noncanonical amino acids (ncAAs). This study reveals a fluorogenic Diels-Alder reaction for super-resolution microscopy and sensitive bioimaging in cells.
Area of Science:
- Biochemistry and Molecular Biology
- Chemical Biology
- Microscopy and Imaging
Background:
- Genetic code expansion (GCE) facilitates the site-specific integration of noncanonical amino acids (ncAAs) into proteins.
- Tetrazine-dye conjugates are known to exhibit fluorescence quenching due to photoinduced electron transfer (PET).
Purpose of the Study:
- To investigate the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs and a panel of tetrazine-dye conjugates.
- To explore the fluorogenic properties of this reaction for bioimaging applications.
- To evaluate the utility of this method for super-resolution microscopy and live-cell imaging.
Main Methods:
- Incorporation of TCO-modified ncAAs into proteins using GCE.
- Reaction of TCO-modified proteins with 22 tetrazine-dye conjugates across the visible spectrum.
- Characterization of the Diels-Alder reaction mechanism, including PET.
- Application of the labeled proteins in super-resolution microscopy and live-cell imaging.
Main Results:
- The Diels-Alder reaction between TCO-ncAAs and tetrazines is fluorogenic, with PET identified as the primary quenching mechanism in oxazine and rhodamine dyes.
- Reaction with dienophiles (TCO-ncAAs) significantly reduces quenching, leading to a substantial increase in fluorescence intensity.
- Efficient and specific protein labeling was achieved across all tested tetrazine-dyes.
- The method enabled super-resolution microscopy with high signal-to-noise ratios, even at the single-molecule level.
- Differential cell permeability of tetrazine-dyes allowed for selective intra- and extracellular protein labeling.
Conclusions:
- The developed fluorogenic Diels-Alder reaction using TCO-ncAAs and tetrazine-dyes is a powerful tool for protein labeling.
- This approach is suitable for high-resolution imaging, including single-molecule and live-cell applications.
- The tunable cell permeability of tetrazine-dyes offers versatility for specific labeling strategies in complex biological systems.
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