Precise Control of Micropipette Flow Rate for Fluorescence Imaging in In Vivo Micromanipulation

Precise regulation of micropipette outlet flow is critical for fluorescence imaging in vivo micromanipulations. In such procedures, a micropipette with a micro-sized opening is driven by gas pressure to deliver internal solution into the in vivo environment. The outlet flow rate needs to be precisely regulated to ensure a uniform and stable fluorescence distribution. However, conventional manual pressure injection methods face inherent limitations, including insufficient precision and poor reproducibility. Existing commercial microinjection systems lack a quantitative relationship between pressure and flow rate. And existing calibration methods in the field of microfluidics suffer from a limited flow-rate measurement resolution, constraining the establishment of a precise pressure–flow quantitative relationship. To address these challenges, we developed a closed-loop pressure regulation system with 1 Pa-level control resolution and established a quantitative calibration of the pressure–flow relationship using a droplet-based method. The calibration revealed a linear relationship with a mean pressure–flow gain of 4.846 $\times {10}^{-17}{\mathrm{m}}^3·{\mathrm{s}}^{-1}·{\mathrm{P}\mathrm{a}}^{-1}$ (R² > 0.99). Validation results demonstrated that the system achieved the target outlet flow rate with a flow control error less than 10 fL/s. Finally, the application results in brain-slice environment confirmed its capability to maintain stable fluorescence imaging, with fluorescence intensity fluctuations around 1.3%. These results demonstrated that the proposed approach provides stable, precise, and reproducible flow regulation under physiologically relevant conditions, thereby offering a valuable tool for in vivo micromanipulation and detection.