Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Understanding microtubule dynamics is crucial for cell division and motility.
• Existing methods for observing microtubule growth can be limited by phototoxicity and artifacts.

Purpose of the Study:

• To describe a modified, high-resolution live-cell imaging protocol for microtubule dynamics.
• To improve the accuracy and reproducibility of microtubule growth analysis in living cells.

Main Methods:

• Utilized EB3 protein labeled with tdTomato fluorescent protein to mark microtubule plus-ends.
• Employed spinning disk confocal microscopy for high-speed, high-resolution live-cell imaging.
• Synchronized cell cycles and increased microtubule density by inhibiting centrosomal separation; analyzed growth using U-Track software.

Main Results:

• Reduced phototoxicity and artifacts by using a red-shifted fluorescent protein and spinning disk microscopy.
• Enabled imaging of more cells under standard culture conditions with reduced laser power and exposure.
• Achieved statistically robust and reproducible results through supervised automatic analysis.

Conclusions:

• The modified protocol offers a more sensitive and reliable method for studying microtubule dynamics.
• This technique facilitates deeper insights into microtubule function in various cellular processes.