Related Experiment Video
Updated: Aug 5, 2026
07:38
Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging
Published on: May 17, 2010
Protein-based, live-cell PAINT microscopy with fluorogenic exchangeable HaloTag ligands
Tong Zhan1, Nels C Gerstner1, Julia G Martin1
1Department of Chemistry, University of California, Berkeley 94720-1460.
Biorxiv : the Preprint Server for Biology
|July 29, 2026
Summary
This study introduces a new method for super-resolution microscopy in living cells. It enables high-resolution imaging and tracking of proteins, advancing live-cell biological research.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Super-resolution microscopy allows imaging beyond the diffraction limit.
- Live-cell imaging presents challenges due to phototoxicity and speed.
- Protein-based PAINT (points accumulation for imaging in nanoscale topography) offers a versatile super-resolution technique.
Purpose of the Study:
- To develop a generalizable method for live-cell super-resolution microscopy.
- To enhance protein-based PAINT with improved fluorophores and labeling strategies.
- To enable simultaneous multicolor imaging and diffusivity mapping in living cells.
Main Methods:
- Developed a live-cell protein-based PAINT method using bis-trifluoromethyl rhodamine (BF) dyes and reversible HaloTag ligands.
- Integrated far-red emitting small molecule dyes for enhanced labeling.
- Combined with photoactivatable fluorescent proteins for two-color imaging and single-molecule diffusivity mapping (SMdM).
Main Results:
- Achieved high-resolution, live-cell super-resolution microscopy using the novel PAINT method.
- Demonstrated successful simultaneous two-color imaging of cellular structures.
- Enabled precise mapping of single-molecule diffusion dynamics within living cells.
Conclusions:
- The developed method provides a versatile and generalizable approach for live-cell super-resolution microscopy.
- This technique significantly advances the ability to study dynamic cellular processes at the nanoscale.
- The combination of BF dyes, HaloTag, and photoactivatable proteins opens new avenues for multicolor super-resolution live-cell imaging and analysis.
Related Concept Videos
03:31Labeling DNA Probes
DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
01:24Tagging and Fusion Proteins
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...