Effect of velocity on microdroplet fluorescence quantified by laser-induced fluorescence

Microdroplets generated inside microfluidic devices have been widely used as miniaturized chemical and biological reactors allowing important reductions in experimental fluid volumes and making it possible to carry out high-throughput assays. Laser-induced fluorescence (LIF) is commonly used to detect and quantify the product, marker or cell content inside each individual droplet. In this work, we employed this technique to characterize the response of in-flow microdroplets filled with fluorescein dye at different laser powers and flow velocities. Using two parallel laser beams closely focused inside a microchannel we determined the microdroplet velocities and showed that the droplet fluorescence intensity decreases exponentially with reducing velocities because of the effects of photobleaching. In contrast, the fluorescence intensity increases linearly with laser power in the 4-10 mW range. When LIF is used for microdroplet measurements it is important to consider not just the fluorophore concentration but also the droplet velocity and laser power in the development of quantitative assays.