Variation of expression with cell size and cell cycle.
A) Fluorescence and cell volume measurements on live cells (Clone 5H6) by imaging flow cytometry. Cell volume inferred by calculation from brightfield projected area (Methods). EGFP and EYFP fluorescence collected in a single channel. Linear fit (red line) and R2 are shown in main panel. Contours are percentiles (5%). Histogram counts on each axis are shown fitted to a log-normal distribution (red curves). Fluorescence in arbitrary units (A.U.). B) Cell image field (montage) illustrating subset of events from (A) with image centers aligned to match corresponding graph coordinates. False-color overlay of bright field and fluorescence channels. Scale bar, 20 µm. Note: brightness is perceived per unit projected area, not per unit volume as plotted, making larger cells appear brighter in cross-section due to depth of field. C) Fixed cells (Clone 5H6) stained with propidium iodide for DNA content and measured by conventional flow cytometry. EGFP fluorescence corrected for cell volume (estimated by FSC-A, see Methods). EYFP similar (not shown). Linear fit (red line) and R2 are shown in main panel. Contours are percentiles (5%). Histogram counts on upper axis indicate cell cycle phases (G0/G1, S, G2/M). Fluorescence in arbitrary units (A.U.).
