Two-color green fluorescent protein time-lapse imaging

Area of Science:

• Cell Biology
• Microscopy
• Biochemistry

Background:

• Green fluorescent protein (GFP) from Aequorea victoria is crucial for tracking proteins and organelles.
• Existing GFP variants often suffer from photobleaching or spectral overlap, limiting their use in dual-labeling, time-lapse studies.
• Improved GFP variants are needed for advanced live-cell imaging applications.

Purpose of the Study:

• To evaluate two novel, photostable spectral green fluorescent protein (GFP) variants, W7 and 10C, for dual-color, time-lapse imaging.
• To demonstrate the suitability of W7 and 10C for live-cell imaging without spectral correction.
• To visualize simultaneous dynamics of different cellular compartments.

Main Methods:

• Developed and utilized two photostable spectral GFP variants, W7 and 10C.
• Performed dual-color, time-lapse imaging of fusion proteins in COS-7 cells using wide-field and confocal microscopy.
• Co-expressed W7-fused Golgi marker (galactosyl transferase) and 10C-fused nuclear envelope marker (lamin-B receptor).

Main Results:

• W7 and 10C exhibited high photostability during extended imaging periods.
• Distinct excitation spectra allowed clean separation of W7 and 10C fluorescence with negligible spectral crossover.
• Successfully generated time-lapse sequences showing simultaneous visualization of Golgi and nuclear envelope membranes in living cells.

Conclusions:

• W7 and 10C are highly effective and photostable spectral GFP variants for dual-color, time-lapse live-cell imaging.
• These variants overcome limitations of previous GFP combinations, enabling simultaneous observation of cellular dynamics.
• This study presents the first simultaneous live-cell imaging of Golgi and nuclear envelope membranes.