Optical measurement of cycle-dependent cell growth

Abstract

Determining the growth patterns of single cells offers answers to some of the most elusive questions in contemporary cell biology: how cell growth is regulated and how cell size distributions are maintained. For example, a linear growth in time implies that there is no regulation required to maintain homeostasis; an exponential pattern indicates the opposite. Recently, there has been great effort to measure single cells using microelectromechanical systems technology, and several important questions have been explored. However, a unified, easy-to-use methodology to measure the growth rate of individual adherent cells of various sizes has been lacking. Here we demonstrate that a newly developed optical interferometric technique, known as spatial light interference microscopy, can measure the cell dry mass of many individual adherent cells in various conditions, over spatial scales from micrometers to millimeters, temporal scales ranging from seconds to days, and cell types ranging from bacteria to mammalian cells. We found evidence of exponential growth in Escherichia coli, which agrees very well with other recent reports. Perhaps most importantly, combining spatial light interference microscopy with fluorescence imaging provides a unique method for studying cell cycle-dependent growth. Thus, by using a fluorescent reporter for the S phase, we measured single cell growth over each phase of the cell cycle in human osteosarcoma U2OS cells and found that the G2 phase exhibits the highest growth rate, which is mass-dependent and can be approximated by an exponential.

Fig. 1.

SLIM measurements of E. coli growth. (A) Dry mass vs. time for a cell family. Growth curves for each cell are indicated by the colored circles on the images. Images show single cell dry mass density maps at the indicated time points (in minutes). (Scale bar: 2 μm.) (Inset) Histogram of the dry mass noise associated with the background of the same projected area as the average cell (SD σ = 1.9 fg is shown). The blue line is a fixed cell measurement, with SD of 19.6 fg. Markers indicate raw data, and solid lines indicate averaged data. (B) Growth rate vs. mass of 20 cells measured in the same manner. Faint circles indicate single data points from individual cell growth curves, dark squares show the average, and the dashed line is a linear fit through the averaged data; the slope of this line, 0.011 min⁻¹, is a measure of the average growth constant for this population. The linear relationship between the growth rate and mass indicates that, on average, E. coli cells exhibit exponential growth behavior.

Fig. 2.

SLIM measurement of synchronized U2OS cell culture over >2 d. Black shows dry mass vs. time for a synchronized cell population over a 3.2 × 2.4-mm² field of view obtained by montaging 8 × 8 microscope images. (10× objective, NA = 0.3). Red shows cell mean dry mass vs. time. Images show the field of view at 4 and 45 h; horizontal edge of image is 2.4 mm.

Fig. 3.

SLIM measurement of U2OS growth over 2 d. (A) Dry mass density maps of a single U2OS cell over its entire cycle at the times indicated. (Scale bar: 25 μm.) Color bar indicates dry mass density in pg/μm². (B) Simultaneously acquired GFP fluorescence images indicating PCNA activity; the distinct GFP signal during S phase and the morphological changes during mitosis allow for determination of the cell cycle phase. (C) Dry mass vs. time for a cell family (i.e., 1→2→4 cells). The two different daughter cell lineages are differentiated by the filled and open markers; only one daughter cell from each parent is shown for clarity. Different colors indicate the cell cycle as reported by the GFP–PCNA fluorescence. The dotted black line shows measurements from a fixed cell, which has SD of 1.02 pg.

Fig. 4.

(A) A posteriori synchronization combination of PCNA stain for S-phase determination and the visual determination of the onset of mitosis allow for the study of cell growth dependence on cell cycle phase in an asynchronous culture. Show is a G1-, S-, and G2-dependent mass growth as indicated by color. The cycles of the individual cells were aligned as described above; the x axis indicates the average time spent in the respective cell cycle phase by all. Open circles indicate single cell data, and solid lines indicate ensemble averages by cell cycle phase. It can clearly be seen that the cell growth is dependent on both the cell cycle phase and the current mass of the cell. (B) Dry mass growth rate vs. dry mass for the ensemble averages. It can be seen that G2 exhibits an exponential growth pattern compared with the relatively low growth measured in G1 and S phases.