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Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy.

Julian Kompa¹, Jorick Bruins¹, Marius Glogger²

¹Department of Chemical Biology, Max Planck Institute for Medical Research, Jahnstrasse 29, Heidelberg 69120, Germany.

Journal of the American Chemical Society

|January 30, 2023

Summary

This summary is machine-generated.

We developed reversible HaloTag ligands (xHTLs) for advanced bioimaging. These exchangeable ligands reduce photobleaching and enable new super-resolution microscopy techniques like PAINT and MINFLUX.

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Area of Science:

Cellular and Molecular Imaging
Biotechnology
Super-resolution Microscopy

Background:

HaloTag protein labeling is crucial for bioimaging in living cells.
Covalent probes irreversibly bind HaloTag, limiting applications and causing photobleaching.

Purpose of the Study:

To introduce exchangeable ligands for HaloTag (xHTLs) enabling reversible, transient binding.
To overcome limitations of irreversible covalent HaloTag labeling for advanced imaging.

Main Methods:

Development of exchangeable ligands for fluorescence labeling of HaloTag (xHTLs).
Coupling of xHTLs to rhodamine dyes for multicolor imaging.
Application of xHTLs in stimulated emission depletion (STED) microscopy, Points Accumulation for Imaging in Nanoscale Topography (PAINT), and MINFLUX microscopy.
Design of xHTL and HaloTag mutant pairs for dual-color super-resolution imaging.

Main Results:

xHTLs exhibit reversible binding to HaloTag, reducing photobleaching in STED microscopy.
Fluorogenic xHTLs facilitate transient binding for PAINT and MINFLUX super-resolution imaging.
Successful demonstration of dual-color PAINT and STED microscopy using xHTLs and HaloTag mutants.

Conclusions:

Exchangeable ligands (xHTLs) offer a versatile alternative to covalent probes for HaloTag labeling.
xHTLs expand imaging possibilities across multiple microscopy platforms, including super-resolution techniques.
This approach enhances bioimaging by enabling transient binding and reducing photobleaching.