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Figure 3: Design and functionality of microfluidic sorting device A) Microfluidic device

guides microscaffolds past detection area to either sort BBs in well plate beneath or direct
towards waste outlet B) Cross-view of pneumatic valves and fluidic conduit of sorting device

C) Sequence overview of sorting procedure. V1 and V2 denote on-chip valves and AI denotes

pressurized air inlet. Colors reflect their state (green = open, red = closed) at different sorting
stages.

Flow focusing was used to arrange the incoming microscaffolds in succession to generate
single events at the fluorescent detection area downstream. With microscaffolds measuring 300

µm in diameter, a sample stream width of around 400 µm was targeted. As seen in Figure 4a-

f, the width of the central sample stream changed by varying the liquid pressure ratio between
sample and sheath liquid. At a 1:1 ratio, the inner channel took up 50% of the channel (~ 400

µm). At a 1:2 ratio, the inner channel was reduced to 30% (~240 µm) and at a 1:3 ratio, it was
further reduced to 20 % (~165 µm). Conversely, at a ratio of 2:1, the channel took up 71%

(~570 µm). At a ratio of 3:1, no further distinction between sample liquid and sheath liquid
could be made, similar to when sheath flow was turned off completely, as quantified and

summarized in Figure 4j. The laminar flow regime in the sorting device was kept intact

regardless of the chosen pressure ratios (Figure 4a-f) and flow speeds (Figure 4g-i).

Once the microscaffolds flowed in succession, their travel speed was measured to determine

the timing offset between BB detection and rerouting back to the ejection channel (Figure 3 a).

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