Controlling gas/liquid exchange using microfluidics for real-time monitoring of flagellar length in living Chlamydomonas at the single-cell level.

Chlamydomonas reinhardtii is widely used for studying cilia/flagella, organelles important for human health and disease. In situ monitoring of flagellar assembly/disassembly kinetics in single living cells has been difficult with conventional methods because of time-consuming media exchange and the requirement of whole cell fixation. Here, we develop a PDMS/glass hybrid microfluidic device for real-time tracking of flagellar length in single living cells of Chlamydomonas. Media exchange is precisely controlled by sequential gas-liquid plugs and complete medium replacement occurs within seconds. Rapid medium exchange allows the capture of transient flagellar dynamics. We show that Chlamydomonas cells respond to acidic medium exchange and deflagellate. However, the two flagella may shed asynchronously. After subsequent medium exchange, cells regenerate full-length flagella. Cells are also induced to shorten their flagella after being exposed to extracellular stimuli. The long-term kinetics of flagellar regeneration and disassembly for the whole cell population on the chip are comparable to those from conventional methods; however, individual cells display non-uniform response kinetics. We also find that flagellar growth rate is dependent on flagellar length. This device provides a potential platform to continuously monitor molecular activities associated with changes in flagellar length and to capture transient molecular changes upon flagellar loss, and initiation of flagellar assembly/disassembly.