Single-Molecule Localization Microscopy in the Nucleus with Self-Labelling Tags: Halo, SNAP, and CLIP Tags.

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Single-molecule localization microscopy (SMLM) of nuclear proteins presents challenges due to the need for brighter, longer-lasting fluorescent tags.
• Fluorescent protein (FP) tags cannot be fused to nuclear proteins, while fluorescent ligands lack cell permeability.
• Self-labelling tags, like HaloTag, offer a solution by combining fusion protein expression with the optical advantages of external fluorescent dyes.

Purpose of the Study:

• To describe protocols for high-resolution SMLM of nuclear proteins within live cells.
• To adapt SMLM techniques for imaging within the confined nuclear environment.
• To utilize self-labelling tags and advanced microscopy for nuclear protein visualization.

Main Methods:

• Cells were seeded and transfected with a HaloTag-tagged nuclear protein of interest.
• Transfected cells were incubated with a fluorescent nuclear marker and a HaloTag ligand.
• Highly Inclined and Laminated Optical sheet (HILO) microscopy with a high-power Total Internal Reflection Fluorescence (TIRF) filter was employed for imaging.

Main Results:

• Successful implementation of protocols for preparing and imaging cells using SMLM.
• Acquisition of high-resolution images of fluorescently labelled nuclear proteins within live cells.
• Demonstration of HILO microscopy's capability for nuclear SMLM with enhanced signal-to-noise ratio.

Conclusions:

• Self-labelling tags combined with HILO microscopy provide an effective approach for nuclear SMLM.
• The described protocols facilitate detailed visualization of nuclear protein dynamics.
• This method advances the study of nuclear protein localization and function at the single-molecule level.