Fig. 2: Spatial hydrodynamic focusing validation and in-focus microparticle DHM detection.

a Schematic representation of vertical focusing (y-z plane) and horizontal focusing (x-y plane). b Experimental results of vertical focusing (y-z plane) and horizontal focusing (x-y plane). The red band represents the sample flow containing fluorescent dye Rhodamine B. The dashed lines represent the boundaries of the microfluidic channels. The sample injection speed is 1 μL/min. R_v represents the velocity ratio of vertical sheath flow to sample flow. R_h represents the velocity ratio of horizontal sheath flow to the combined flows of two vertical sheath flows and sample flow. For the horizontal focusing test, R_v is fixed at 0.25. Each focused sample flow height (H_s) and width (W_s) is the mean of 5 independent repeated experiments under the corresponding R_v and R_h conditions. c Statistic results of spatial hydrodynamic focusing. It shows the change in H_s with R_v, and W_s with R_h. Data (red dots) are mean values from 5 independent repeated experiments; error bars are SD. d, e show the flow observation of 10 μm (d) and 20 μm (e) polystyrene microspheres in microfluidic channels under no focusing, planar focusing, and complete spatial focusing conditions in white light illumination. f, g show the DHM reconstructed amplitude (top) and phase (bottom) maps of 10 μm (f) and 20 μm (g) microspheres under the spatial focusing. The color bar denotes the scale of phase. In (d–g), each microsphere constitutes an independent experimental replicate, and finally, amplitude and phase data are collected from 50 independently captured microspheres under each focusing condition for further analysis. h, i show the reconstructed particle size of 10 μm (h) and 20 μm (i) microspheres under different focusing conditions from amplitude. j, k show the reconstructed particle size of 10 μm (j) and 20 μm (k) microspheres under different focusing conditions from phase. l, m show the distance from particle center to channel centerline of 10 μm (l) and 20 μm (m) microspheres under different focusing conditions. In (h–m), data are presented as means ± SD (n = 50 microspheres under each focusing condition); the p-value < 0.05 indicates a significant difference decided by one-way ANOVA followed by Tukey’s multiple comparisons test. Scale bars: 30 μm (b, y-z plane), 100 μm (b, x-y plane), 50 μm (d), 50 μm (e), 10 μm (f), 10 μm (g). Source data are provided as a Source Data file.