A highly stable monomeric red fluorescent protein for advanced microscopy.

Area of Science:

• Biophysics
• Molecular Biology
• Microscopy

Background:

• Fluorescent proteins (FPs) are essential tools for biological imaging.
• Stable green and yellow FPs are available, but stable monomeric red FPs (RFPs) are limited.
• This limits advanced imaging applications requiring red fluorescence.

Purpose of the Study:

• To develop an extremely stable monomeric red fluorescent protein (RFP).
• To characterize its structure and performance in various microscopy techniques.
• To establish mScarlet3-H as a superior RFP for multimodality imaging.

Main Methods:

• Development and structural determination (1.5 Å resolution) of a novel monomeric RFP, mScarlet3-H.
• Assessment of mScarlet3-H stability under thermal, chaotropic, and oxidative stress.
• Benchmarking mScarlet3-H performance in live-cell imaging, super-resolution microscopy, and tissue clearing.

Main Results:

• mScarlet3-H demonstrates exceptional stability against various stressors.
• It enables rapid whole-organ tissue clearing and long-term 3D structured illumination microscopy.
• High photostability and signal-to-noise ratio facilitate dual-color live-cell super-resolution imaging.

Conclusions:

• mScarlet3-H is a highly stable monomeric RFP suitable for demanding imaging applications.
• It significantly advances correlative light and electron microscopy and live-cell imaging.
• mScarlet3-H complements green FPs for multiplexed imaging in diverse biological systems.