Extended Data Fig. 2: Determination of bead sizes using RT-FDC, soRT-FDC, and flow cytometry (FC).
From: Intelligent image-based deformation-assisted cell sorting with molecular specificity
A mixture of beads of four different sizes, each made of different material, was analyzed with (so)RT-FDC and FC (BD LSR II, BD Biosciences). We gated for the different bead populations using two-dimensional analysis combining size and brightness in RT-FDC (a) and soRT-FDC (b), and forward scatter area (FSC-A) and side scatter area (SSC-A) in FC (c). In a–c color map in scatter plots represents event density, the histograms of area and FSC-A are shown on top of scatter plots, and the histograms of brightness and SSC-A on the right-hand side of the scatter plots. Color-coded solid lines represent Gaussian fits to the gated populations of beads. For illustration purposes, data in a–c were resampled to equalize the relative content of each bead population. (d) A summary of bead properties, including bead material, and bead sizes as specified by the supplier, measured with RT-FDC (using 40 × objective and glass substrate) and soRT-FDC (using 20× objective, lithium niobate substrate and an extra polarizer), as well as measured with FC. The number of events, n, analyzed for each bead type using respective methods is indicated in the table. Note that the bead size determined by FC is a dimensionless value and not a physical dimension. Furthermore, the relative bead sizes determined with FC do not follow the size order specified by the supplier, as indicated by increasing grey levels in the background. The experiments in a–c were repeated independently 2, 3, and 1 times, respectively, with similar results.
