This work introduces three rectangular quasi-flat-top spots, which are provided by binary optical elements (BOEs) and utilized for the illumination in a microflow cytometer. The three spots contain, respectively, one, two, and three rectangles (R1, R2, and R3). To test the performance of this mechanism, a microflow cytometer is established by integrating the BOEs and a three-dimensional hydrodynamic focusing chip. Through the experiments of detecting fluorescence microbeads, the three spots present good fluorescence coefficients of variation in comparison with those derived from commercial instruments. Benefiting from a high spatial resolution, when using R1 spot, the micro flow cytometer can perform a throughput as high as 20 000 events per second (eps). Illuminated by R2 or R3 spot, one bead emits fluorescence twice or thrice, thus the velocity can be measured in real time. Besides, the R3 spot provides a long-time exposure, which is conducive to improving fluorescence intensity and the measurement stability. In brief, using the spots shaped and homogenized by BOEs for illumination can increase the performance and the functionality of a micro flow cytometer.
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