In this work, manipulating width and equilibrium position of fluorescent microparticles in spiral microchannel fractionation devices by embedding microchambers along the last turn of a spiral is reported. Microchambers with different shapes and sizes were tested at Reynolds numbers between 15.7 and 156.6 (100–1000 μL min ~(?1) ) to observe focusing of 2, 5 and 10 μm fluorescent microparticles. This paper also discusses the fabrication process of the microfluidic chips with femtosecond laser ablation on glass wafers, as well as a particle imaging velocimetry (μPIV) study of microparticle trajectories inside a microchamber. It could be demonstrated with an improved final design with inclined microchamber side walls, that the 2 μm particle equilibrium position is shifted towards the inner wall by ～27 μm and the focusing line's width is reduced by ～18 μm. Finally, Saccharomyces cerevisiae yeast cells were tested in the final chip and a cell focusing efficiency of 99.1% is achieved.
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