A micro flow cytometer with a single step 3D hydrodynamic flow focusing has been developed. The proposed design is capable to create a single-file particle stream that is self-aligned with an integrated optical fiber-based detection system, regardless of the flow rate ratio between the focusing and core liquids. The design approach provides the ability to adjust the stream size while keeping the position of the focused stream centered with respect to the focusing channel. The device has been fabricated by direct micro milling of PMMA sheets. Experimental validation of the hydrodynamic sheath focusing effect has been presented and sample stream with tuneable size from about 18 to 50 μm was measured. Flow cytometry measurements have been performed by using 10-23 μm fluorescent particles. From the analysis of the signals collected at each transit event we can confirm that the device was capable to align and measure microparticles with a good coefficient of variance.
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