Fig. 2

Evaluating the Precision and Accuracy of Quantitative Microscopy with Calibration Beads. A Example images of uniformly fluorescent spectrum calibration beads. B Triplicate evaluations of beads imaged at different focal planes show fluorescence intensity is significantly different at a 2 μm z-plane differential. Each symbol represents the average of the log transformed mean fluorescence intensities (MFI) of all beads within each large image (7 × 7 mm area, ~ 10,000 beads per image). C Beads imaged and quantified by three different analysts demonstrate the high precision of the workflows (%CV values annotated on graphs). D Schematic outlining how antibody-coated polymer beads (8 uM diameter) can mimic single-cells expressing different quantities of proteins, enabling an evaluation of accuracy. E Image of a bead showing internal dye used to define the binary layer boundary, and the fluorescence signal associated with the fluorescent anti-PD-L1 detection antibody binding to the PD-L1 ligand. F Serial dilution of PD-L1 ligand and G HLA I ligand demonstrate the accuracy of the image acquisition and analytical workflows to quantify protein expression (each symbol represents the average logMFI of all beads quantified from one large 7 × 7 mm image from each condition (~ 1000 beads per image))