Long-Term Imaging of DNA Damage and Cell Cycle Progression in Budding Yeast Using Spinning Disk Confocal Microscopy

Area of Science:

• Cell Biology
• Microscopy Techniques
• Genetics

Background:

• Live cell imaging is crucial for studying cellular processes but illumination can perturb sensitive yeast cells.
• Yeast cells are small, photosensitive, and have low protein content, making them particularly susceptible to imaging-induced damage.
• Analyzing the DNA damage response (DDR) requires methods that avoid introducing artifacts.

Purpose of the Study:

• To provide a guide and resources for imaging the DNA damage response (DDR) in Saccharomyces cerevisiae over extended durations (3-12 hours).
• To establish a microscopy setup that minimizes photobleaching and photodamage, crucial for studying yeast cell physiology.
• To facilitate high-resolution, time-lapse imaging of DDR activation and its impact on cell cycle progression.

Main Methods:

• Utilized spinning-disk confocal microscopy for live cell imaging of yeast.
• Developed specific strains and imaging protocols tailored for S. cerevisiae.
• Employed conditions designed to limit photobleaching and photodamage during extended time-lapse recordings.

Main Results:

• Successfully imaged the DNA damage response (DDR) in S. cerevisiae for 3-12 hours.
• Demonstrated a method that minimizes light-induced artifacts, preserving cellular physiology.
• Enabled detailed observation of DDR dynamics and consequences for cell cycle progression.

Conclusions:

• The presented spinning-disk confocal microscopy approach is effective for long-term, low-damage live cell imaging of DDR in yeast.
• This method is valuable for studying genomic stability, cell cycle control, and the effects of mutations or drugs.
• The guide and strains facilitate research into DNA repair mechanisms and cellular stress responses in yeast.