Continuous flow technology for particle focusing in microscopy

Imaging flow cytometry provides access to high-throughput imaging of bioparticles in continuous flow. High-quality images with maximum contrast and optical resolution can be obtained if the particles pass through the flow cell exactly within the focal plane of the imaging system. Microfluidic technologies enable particle focusing along a line or within a horizontal plane. This work explores the potential applications of hydrodynamic flow focusing and acoustofluidic particle focusing as complementary techniques for imaging flow cytometry (IFC). Therefore, the quality of particle focusing has been measured and analyzed in 3D by astigmatic microparticle imaging. In our 3D measuring system, a z-resolution of 10 µm was achieved with a measuring range of +/- 80 µm relative to the focal plane. A simple, but highly efficient method for z-calibration has been proposed and validated. Ultimately, both flow focusing approaches enable precise z-focusing of 10 µm-sized test particles into narrow lamellas with a standard deviation of 3 µm in the z-direction. At the same time, they cover complementary use cases, so that both methods are justified in their application.