LIVE-PAINT allows super-resolution microscopy inside living cells using reversible peptide-protein interactions

Area of Science:

• Cellular and Molecular Imaging
• Biophysics
• Microscopy Techniques

Background:

• Conventional super-resolution microscopy often requires direct fusion of the target biomolecule to large fluorescent labels.
• These labels can perturb protein function and limit imaging duration in live cells.
• There is a need for minimally invasive super-resolution techniques for live-cell imaging.

Purpose of the Study:

• To introduce LIVE-PAINT, a novel approach for super-resolution fluorescent imaging in live cells.
• To demonstrate the applicability and advantages of LIVE-PAINT compared to existing methods.
• To enable minimally perturbing, long-duration super-resolution imaging of proteins.

Main Methods:

• LIVE-PAINT utilizes a short peptide sequence fused to the protein of interest.
• It observes reversible binding of the peptide to a fluorescently tagged protein, inducing localized fluorescence blinking.
• The method was tested for imaging various proteins in live Saccharomyces cerevisiae.

Main Results:

• LIVE-PAINT successfully achieved super-resolution imaging of proteins in live yeast cells.
• The peptide-based approach proved widely applicable and easy to implement.
• Modifications were minimally perturbing to cellular processes.
• Extended data acquisition times were anticipated compared to direct fusion methods.

Conclusions:

• LIVE-PAINT offers a versatile and minimally invasive method for live-cell super-resolution imaging.
• The technique overcomes limitations of traditional fluorescent protein fusions.
• LIVE-PAINT facilitates longer observation times for dynamic cellular processes at super-resolution.