Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging

Area of Science:

• Biotechnology
• Molecular Biology
• Microscopy

Background:

• Far-red fluorescent proteins (FPs) are vital for live imaging but require optimization.
• Existing far-red markers often lag behind green fluorescent protein (GFP) variants in performance.

Purpose of the Study:

• To introduce mGarnet, a novel monomeric far-red fluorescent protein.
• To characterize mGarnet's photophysical properties and assess its utility in advanced microscopy.

Main Methods:

• Spectroscopic analysis to determine extinction coefficient, excitation/emission peaks, and quantum yield.
• Demonstration of mGarnet as a live-cell fusion marker using STED microscopy.

Main Results:

• mGarnet exhibits far-red fluorescence peaking at 670 nm with a high extinction coefficient (95,000 M⁻¹cm⁻¹).
• It shows efficient excitation at 640 nm and a large Stokes shift.
• mGarnet demonstrated excellent performance in live-cell fusion assays with STED microscopy.

Conclusions:

• mGarnet is a robust monomeric far-red marker protein suitable for advanced live-cell imaging.
• Its properties, including a large extinction coefficient and Stokes shift, compensate for typical far-red quantum yields.
• mGarnet represents a significant advancement for far-red fluorescence imaging applications.