We used a suspended microchannel resonator (SMR) combined with picoliter-scale microfluidic control to measure buoyant mass and determine the ‘instantaneous’ growth rates of individual cells. The SMR measures mass with femtogram precision, allowing rapid determination of the growth rate in a fraction of a complete cell cycle. We found that for individual cells of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and mouse lymphoblasts, heavier cells grew faster than lighter cells.

We have used a microfluidic mass sensor to measure the density of single living cells. By weighing each cell in two fluids of different densities, our technique measures the single-cell mass, volume, and density of approximately 500 cells per hour with a density precision of 0.001 g mL −1 . We observe that the intrinsic cell-to-cell variation in density is nearly 100-fold smaller than the mass or volume variation. As a result, we can measure changes in cell density indicative of cellular processes that would be otherwise undetectable by mass or volume measurements. Here, we demonstrate this with four examples: identifying Plasmodium falciparum malariainfected erythrocytes in a culture, distinguishing transfused blood cells from a patient's own blood, identifying irreversibly sickled cells in a sickle cell patient, and identifying leukemia cells in the early stages of responding to a drug treatment. These demonstrations suggest that the ability to measure single-cell density will provide valuable insights into cell state for a wide range of biological processes.cell size | apoptosis | hematology | biosensor | suspended microchannel resonator

Cell growth comprises changes in both mass and volume-two processes that are distinct, yet coordinated through the cell cycle. Understanding this relationship requires a means for measuring each of the cell's three basic physical parameters: mass, volume, and the ratio of the two, density. The suspended microchannel resonator weighs single cells with a precision in mass of 0.1% for yeast. Here we use the suspended microchannel resonator with a Coulter counter to measure the mass, volume, and density of budding yeast cells through the cell cycle. We observe that cell density increases prior to bud formation at the G1/S transition, which is consistent with previous measurements using density gradient centrifugation. To investigate the origin of this density increase, we monitor relative density changes of growing yeast cells. We find that the density increase requires energy, function of the protein synthesis regulator target of rapamycin, passage through START (commitment to cell division), and an intact actin cytoskeleton. Although we focus on basic cell cycle questions in yeast, our techniques are suitable for most nonadherent cells and subcellular particles to characterize cell growth in a variety of applications.biosensor | cell growth | cell size | microfluidics | Saccharomyces

Cell size, measured as either volume or mass, is a fundamental indicator of cell state. Far more tightly regulated than size is density, the ratio between mass and volume, which can be used to distinguish between cell populations even when volume and mass appear to remain constant. Here we expand upon a previous method for measuring cell density involving a suspended microchannel resonator (SMR). We introduce a new device, the dual SMR, as a high-precision instrument for measuring single-cell mass, volume, and density using two resonators connected by a serpentine fluidic channel. The dual SMR designs considered herein demonstrate the critical role of channel geometry in ensuring proper mixing and damping of pressure fluctuations in microfluidic systems designed for precision measurement. We use the dual SMR to compare the physical properties of two well-known cancer cell lines: human lung cancer cell H1650 and mouse lymphoblastic leukemia cell line L1210.

We demonstrate the measurement of mass, density, and size of cells and nanoparticles using suspended microchannel resonators. The masses of individual particles are quantified as transient frequency shifts, while the particles transit a microfluidic channel embedded in the resonating cantilever. Mass histograms resulting from these data reveal the distribution of a population of heterogeneously sized particles. Particle density is inferred from measurements made in different carrier fluids since the frequency shift for a particle is proportional to the mass difference relative to the displaced solution. We have characterized the density of polystyrene particles, Escherichia coli, and human red blood cells with a resolution down to 10 −4 g/cm 3 .

An intrinsic uncertainty in particle mass sensing with the suspended microchannel resonator results from variation in a particle's position near the free end of the resonator. To circumvent this error we employ the second flexural bending mode. This mode exhibits additional frequency peaks while particles pass over the antinode, a point where the frequency shift is insensitive to the lateral position of the particle. We measure polystyrene beads with the first and second modes and confirm that the second mode sensing provides a narrower mass histogram. For 3 μm diameter beads, second mode sensing at the antinode improves the coefficient of variation in buoyant mass from 1.76% to 1.05% for population measurements and from 1.40% to 0.53% for a single trapped particle.

We demonstrate a method to enhance the time resolution of a commercial Coulter counter and enable continuous and long-term cell size measurements for growth rate analyses essential to understanding basic cellular processes, such as cell size regulation and cell cycle progression. Our simple modifications to a commercial Coulter counter create controllable cell culture conditions within the sample compartment and combine temperature control with necessary adaptations to achieve measurement stability over several hours. We also wrote custom software, detailed here, to analyze instrument data files collected by either this continuous method or standard, periodic sampling. We use the continuous method to measure the growth rate of yeast in G1 during a prolonged arrest and, in different samples, the dependency of growth rate on cell size and cell cycle position in arrested and proliferating cells. We also quantify with high time resolution the response of mouse lymphoblast cell culture to drug treatment. This method provides a technique for continuous measurement of cell size that is applicable to a large variety of cell types and greatly expands the set of analysis tools available for the Coulter counter.

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