Lifetime-based photoconversion of EGFP as a tool for FLIM

Area of Science:

• Biochemistry
• Cell Biology
• Microscopy

Background:

• Enhanced Green Fluorescent Protein (EGFP) is widely used in biological research, yet some photochemical properties remain unexplored.
• Existing EGFP applications do not fully leverage its potential for advanced imaging techniques.

Purpose of the Study:

• To investigate and characterize the previously undocumented lifetime photoconversion of EGFP.
• To explore the utility of this photoconversion in fluorescence lifetime imaging (FLIM) and protein tracking.

Main Methods:

• Utilized fluorescence lifetime imaging and spectral FLIM to analyze EGFP photoconversion.
• Irradiated EGFP with intense blue light to induce photoconversion and characterized the resulting PC-EGFP properties.

Main Results:

• Demonstrated permanent photoconversion of EGFP to a short-lifetime form (PC-EGFP) using blue light.
• PC-EGFP shows blue-shifted absorption, pH-independent fluorescence, and is not reversible with 405 nm light.
• Successfully tracked nucleophosmin mutant relocalization in live cells using EGFP photoconversion.

Conclusions:

• Controlled EGFP photoconversion offers a valuable tool for kinetic FLIM applications, distinct from spectral channels.
• PC-EGFP can be differentiated from unconverted EGFP via time-resolved detection, minimizing FLIM artifacts.
• This lifetime photoconversion capability positions EGFP advantageously for FLIM, addressing a scarcity of suitable labels.