An optimal three-dimensional focusing technique for micro-flow cytometers

This study presents a novel three-dimensional (3-D) hydrodynamic focusing technique for micro-flow cytometers. In the proposed approach, the sample stream is initially compressed in the horizontal direction by two sheath flows such that it is constrained in the central region of the microchannel. The sample stream is then focused in the vertical direction by a second pair of sheath flows and subsequently passes over a micro-weir structure positioned directly beneath an optical detection system. The microchannel configuration and operational parameters are optimized by performing a series of numerical simulations to examine the effects on the sample stream distribution of the vertical and horizontal focusing ratios, the entrance angle of the second set of sheath flow channels, and the width and depth of the second set of sheath flow channels. The results indicate that the horizontal and vertical sheath flows successfully constrain the sample stream within a narrow, well-defined region of the microchannel. Furthermore, the micro-weir structure results in the separation of the cells/particles in the vertical direction and ensures that they flow in a sequential fashion through the detection region of the microchannel and can therefore be reliably counted. It is shown that the 3-D focusing technique can achieve a focused sample stream width of between 6 and 15 mu m given an appropriate value of the horizontal focusing ratio. Thus, the viability of the microflow cytometer for the counting and detection of individual biological cells is confirmed.