Measuring fast gene dynamics in single cells with time-lapse luminescence microscopy

Area of Science:

• Biophysics
• Molecular Biology
• Cell Biology

Background:

• Time-lapse fluorescence microscopy is crucial for studying gene dynamics in single cells.
• Fluorescent proteins have limitations including slow maturation, autofluorescence, and phototoxicity.
• Luciferase offers an alternative with photon emission upon folding, but typically has lower photon flux.

Purpose of the Study:

• To evaluate beetle luciferases as a superior alternative to fluorescent proteins for in vivo gene expression studies.
• To optimize substrate conditions for enhanced in vivo luminescence.
• To develop a high-resolution, time-lapse luminescence microscopy method for tracking gene dynamics in single yeast cells.

Main Methods:

• Tested green, yellow, and red beetle luciferases for in vivo luminescence.
• Optimized substrate conditions for luminescence detection.
• Integrated time-lapse luminescence microscopy with microfluidic devices for single-cell analysis in yeast.
• Achieved subminute exposure times for tracking gene dynamics over multiple generations.

Main Results:

• Demonstrated that luciferase reporters enable faster and more sensitive tracking of gene dynamics compared to fluorescent proteins.
• Observed a 15-20 minute lag in Venus fluorescence compared to luminescence, confirming luciferase's real-time reporting.
• Successfully tracked cell cycle gene dynamics in single yeast cells with high temporal resolution.

Conclusions:

• Luciferase reporters provide a more faithful and rapid readout of gene expression than fluorescent proteins in single-cell studies.
• The developed luminescence microscopy technique overcomes previous limitations, enabling high-resolution dynamic studies in small cell volumes.
• This approach significantly advances the ability to study gene expression dynamics at the single-cell level.