StayGold photostability under different illumination modes

Area of Science:

• Cellular and Molecular Imaging
• Biophotonics
• Microscopy Techniques

Background:

• Fluorescent proteins (FPs) are crucial tools in live cell imaging.
• Photostability is a key limitation for many FPs, especially under intense illumination.
• Existing green-emitting FPs like EGFP, mClover3, and mNeonGreen have varying photostability profiles.

Purpose of the Study:

• To evaluate the photostability of a novel bright fluorescent protein, StayGold.
• To compare StayGold's photostability against established green FPs (EGFP, mClover3, mNeonGreen) across different microscopy illumination modes.
• To investigate the impact of laser scanning confocal microscopy (LSCM) parameters on FP photostability.

Main Methods:

• Expression of StayGold, EGFP, mClover3, and mNeonGreen as histone 2B fusions in HeLa cells.
• Photostability assessment under widefield, structured illumination, and various single-beam and multi-beam laser scanning confocal microscopy (LSCM) illumination modes.
• Systematic examination of single-beam LSCM scanning patterns and their effect on FP photobleaching.

Main Results:

• StayGold demonstrated over one order of magnitude greater photostability than EGFP, mClover3, and mNeonGreen under widefield and structured illumination microscopy.
• StayGold's photostability was significantly reduced in single-beam LSCM compared to multi-beam LSCM.
• FP photostability, including StayGold's, was found to be sensitive to chemical fixation methods.

Conclusions:

• StayGold represents a significant advancement in fluorescent protein photostability for live cell imaging applications.
• Optimizing LSCM scanning patterns is critical for maximizing the utility of photostable FPs like StayGold.
• Further research is needed to fully understand the photobleaching mechanisms and fixation sensitivity of StayGold.