a Technical drawing of glass cuvette and PDMS chip to scale for comparison. Arrows indicate length of constrictions of 2 cm for the cuvette and 300 µm for the PDMS device. b FEM simulations of concentration distribution for the cross-section of the cuvette performed for sample 114 µM MC at Qsa = 200 nl s−1 and sheath 5 mM PEG40000 at Qsh = 1000 nl s−1 yielding a 80 µm virtual channel (left). Arrows indicate the stress \(\sigma _i\) due to the viscosity mismatch at the interface. Adjusting the flow rates to Qsh = 50 nl s−1 while keeping Qsa constant, enables increasing the diameter of the constriction to 260 µm (right). c Representative image of a HL60 cell in a virtual channel of 88 µm diameter (sample 114 µM MC at Qsa = 200 nl s−1, sheath 5 mM PEG40000 at Qsh = 1000 nl s−1, top) and inside a virtual channel of 14 µm diameter (sample 114 µM MC at Qsa = 15 nl s−1, sheath 5 mM PEG40000 at Qsh = 1000 nl s−1, bottom). Projected line plots at top and bottom indicate the squared intensity gradient (arb. units) perpendicular to the flow direction while center between the maxima identify virtual channel interfaces (white dashed lines). Recording of cells and spheroids inside virtual channels has been repeated six times where a fluctuation in virtual channel size of 10% has been observed.