Intracellular water exchange for measuring the dry mass, water mass and changes in chemical composition of living cells

Abstract

We present a method for direct non-optical quantification of dry mass, dry density and water mass of single living cells in suspension. Dry mass and dry density are obtained simultaneously by measuring a cell's buoyant mass sequentially in an H2O-based fluid and a D2O-based fluid. Rapid exchange of intracellular H2O for D2O renders the cell's water content neutrally buoyant in both measurements, and thus the paired measurements yield the mass and density of the cell's dry material alone. Utilizing this same property of rapid water exchange, we also demonstrate the quantification of intracellular water mass. In a population of E. coli, we paired these measurements to estimate the percent dry weight by mass and volume. We then focused on cellular dry density - the average density of all cellular biomolecules, weighted by their relative abundances. Given that densities vary across biomolecule types (RNA, DNA, protein), we investigated whether we could detect changes in biomolecular composition in bacteria, fungi, and mammalian cells. In E. coli, and S. cerevisiae, dry density increases from stationary to exponential phase, consistent with previously known increases in the RNA/protein ratio from up-regulated ribosome production. For mammalian cells, changes in growth conditions cause substantial shifts in dry density, suggesting concurrent changes in the protein, nucleic acid and lipid content of the cell.

Figure 1. Buoyancy of a cell in fluids of different densities and membrane permeabilities.

a) In an H₂O or D₂O based fluid (1 or 3), the cell sinks as a result of the dry content’s density being higher than the surrounding fluid. In a dense impermeable fluid (2), the buoyancy of the cell’s water content dominates and the cell floats. b) The pairing of the different buoyant mass measurements allows the determination of different biophysical parameters of the cell as shown in the plot (not to scale). c) Kernel density estimates of probability densities for dry mass, water mass and total mass of a sample of fixed stationary-phase E. coli. Functions were rescaled so that their maxima were one. Solid bars represent sample medians.

Figure 2. Dry density and dry mass of a bacterial culture.

a) Growth curves of E. coli cultures. A culture was grown for 24 hours, diluted 1000-fold, and allowed to grow again for 24 hours. Samples from the cultures were fixed for analysis at the colored time-points. Solid line is the fit to logistic growth model. b) The dry density of the culture by sampling time point. Technical replicates of these fixed samples show that the changes in density are reproducible and not attributable to instrument error. c) Probability distributions of dry mass, rescaled so that the modal mass had a density of one. Lines of the same color are technical replicates, measured several days apart.

Figure 3. Dry density and dry mass of a yeast culture.

a) Growth curve of a culture started from a 1000-fold dilution of a recently-saturated culture (time 0h). b) Distributions of dry densities for the time points indicated in a). Distributions expected due purely to measurement error (see text) are shown as black dashed lines. c) Dry mass distributions for the same time points. d) Single-cell data for time point 8h. Solid lines is median dry density and dashed lines are 99% bounds on the expected dry densities if all cells actually had the median dry density, given known measurement error (see Methods). e) Dry density distribution medians for several replicates: curves 1 and 2 are technical replicates and 2–4 are biological replicates; curve 3 is for data show in b).

Figure 4. Dry density and mass of proliferating and non-proliferating mammalian cells.

Solid lines are median dry densities and dashed lines are 99% bounds on the expected dry densities if all cells actually had the median dry density, given known measurement error. a) Confluent and proliferating (75% confluency) mouse embryonic fibroblasts. b) Cycloheximide-treated and proliferating L1210 cells. c) IL-3-depleted and proliferating FL5.12 cells. d) Naïve and activated OT-1 T cells.

Figure 5. Dry density and mass of red blood cells.

a) Single-cell dry density and dry mass distributions of two different human erythrocyte samples. Solid and dashed lines of same color indicate technical replicates. Dashed black line is a 99% bound on the expected dry density of a representative sample if all cells had the median dry density, given known measurement error. inset Population mean values from four patient samples. Error bars are standard deviation of the population. b) Comparison to hemoglobin mass per cell determined with an Advia instrument. Dashed line indicates y = x and solid line is a total least squares fit.