Thermally controlled microdroplet formation generated by fatty acid oil.

Microdroplet technology is a cornerstone of modern biophysical analysis for applications such as single-cell studies, drug screening, and chemical synthesis. Many of these applications involve biological and chemical processes that require precise temperature control under physiologically relevant conditions. However, temperature not only influences these processes but also affects droplet formation by altering fluid properties, thereby changing droplet size and encapsulation efficiency. Despite growing interest in environmentally sustainable alternatives to fluorinated oils, the thermal behavior of such systems remains poorly understood. In this study, we introduce a novel experimental approach to investigate thermally controlled microdroplet generation using emu oil as a biocompatible continuous phase. By independently controlling the temperatures of the dispersed and continuous phases, we distinguish their individual contributions to droplet formation. The resulting changes in droplet morphology were quantitatively characterized across a thermal gradient, showing the distinct influence of phase-specific temperature control on droplet generation. Increasing temperature resulted in larger droplets and accelerated the transition from the squeezing to the jetting regime These findings provide practical guidance for temperature-sensitive microfluidic assays, where the aqueous sample and carrier oil are often prepared at different temperatures, and can improve the reproducibility of applications in different fields requiring precise thermal regulation.