Fig. 2: Effects of projection and diffraction on cell size estimation.

a Synthetic images of a stained cell membrane demonstrate the independent and combined effects of diffraction and projection on 2D image formation (scale bar = 1 µm). Diffraction effects were simulated using the experimentally measured instrumental PSF (iPSF) of our imaging system. b Radial profiles of intensity (across the cell width) from each panel from a are compared to show the relative shifts caused by projection and diffraction effects. The black dotted line indicates the true position of the cell boundary. c Radial profiles of synthetic-cell images with two wavelengths of iPSFs are shown. Lower wavelengths (green) cause smaller shifts, as expected from a smaller size of the PSF. d Example images of a cell stained with fluorescent d-amino acids HADA and RADA are shown. As expected, intensity traces across the cell’s width show RADA (red) emission is more diffracted than HADA (blue) emission, and the diffraction is biassed towards the centre of the cell. e A plot showing the average measured width of a population of cells stained with HADA and RADA (error bar = 99% CI). Inter-peak distances from radial profiles of RADA images consistently underestimate the width more than HADA images. f Comparison of radial profiles before and after deconvolution shows that deconvolution does not shift and correct the peak position; it only makes the profile sharper. g Synthetic images of a digital cell, uniformly filled with fluorescence emitters, show the effects of diffraction and projection on 2D image formation (scale bar = 1 µm). h We compare the radial intensity profile (across the cell width) with and without projection and diffraction effects corresponding to the panels in g. The black dotted line indicates the true position of the cell boundary. i Trendlines from synthetic data show that the observed/true width ratio is dependent on the cell width, with the error growing rapidly for narrow cells. The trends, however, occur in opposing directions for membrane-stained cells and cytoplasm-labelled cells. Estimated widths are calculated from the interpeak distance in membrane-stained cells and full width at half maximum (FWHM) of the radial profile of cytoplasm-labelled cells.