Fig. 4: Colony size affects single-cell intensity quantification—real and synthetic data.

a The radial intensity profile of a simulated fluorescence cell illustrates how intensity is lost from the cell contours due to diffraction effects (scale bar = 0.5 μm). b Example snapshots of simulated fluorescence images of a growing microcolony using a 100× 1.49 NA objective’s instrumental point spread function (iPSF). All cells in the simulation have identical ground truth intensities. Isolated cells and individual cells in small microcolony sizes show low cell intensities, while as the colony grows larger, cells in dense regions, such as at the centre of the colony, begin receiving more light contributions from surrounding cells, artificially increasing their perceived intensity. (Scale bar = 2 μm). c As colony size increases, the mean observed intensity of each cell in the colony also increases (Error bars = 99% CI). Relative changes in intensity compared to isolated individual cells are shown on the left-hand y-axis. Cells approach the true mean intensity as the colony size increases, as shown on the right-hand y-axis. d A false-coloured image of cells on a real agarose pad showing ‘preformed’ microcolonies of various sizes, along with a single cell (white arrow). The relative intensity scale is shown on the right. Important features to note are the similarity to the simulated data shown in (b) (Scale bar = 10 μm). e The intensity distribution of cells depends on the size of the clusters they belong to. Isolated individual cells have low mean intensities, while cells from preformed microcolonies with more than 50 cells have 3× higher mean intensities. f Experimental data shows that the average intensity of cells increases with the population size of microcolonies (shown in orange). The trend from simulated colonies is shown in blue.