Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability.

Area of Science:

• Biophotonics
• Cell Biology
• Microfluidics

Background:

• Mammalian cell selection often requires time-consuming methods.
• Photobleaching of fluorescent proteins is a key factor in cellular imaging and analysis.
• Developing rapid, high-purity cell sorting techniques is crucial for biological research.

Purpose of the Study:

• To develop a novel microfluidic cytometer for rapid, high-purity selection of individual mammalian cells.
• To quantify irreversible photobleaching of red fluorescent proteins in single cells.
• To enable cell sorting based on photostability for subsequent research.

Main Methods:

• Utilized a microfluidic chip for cell handling and analysis.
• Employed a series of 532-nm laser beams to induce photobleaching.
• Used sub-millisecond timed control of a piezo-tilt mirror with a 1064-nm laser for optical gradient force switching.
• Differentiated between reversible dark-state conversion and irreversible photobleaching.

Main Results:

• Achieved high-purity (>99%) selection of individual mammalian cells.
• Demonstrated rapid measurement of irreversible red fluorescent protein photobleaching.
• Enabled sorting of sub-millilitre volumes with minimal cell loss.
• Confirmed collected cells remained viable and capable of proliferation.

Conclusions:

• The developed microfluidic cytometer is a unique tool for rapid, individual mammalian cell selection based on photostability.
• This platform facilitates the development of advanced red fluorescent proteins through genetic library screening.
• The technology lays the groundwork for future lab-on-a-chip platforms integrating photobleaching with other spectroscopic measurements.