High-throughput ballistic injection nanorheology to measure cell mechanics

Abstract

High-throughput ballistic injection nanorheology is a method for the quantitative study of cell mechanics. Cell mechanics are measured by ballistic injection of submicron particles into the cytoplasm of living cells and tracking the spontaneous displacement of the particles at high spatial resolution. The trajectories of the cytoplasm-embedded particles are transformed into mean-squared displacements, which are subsequently transformed into frequency-dependent viscoelastic moduli and time-dependent creep compliance of the cytoplasm. This method allows for the study of a wide range of cellular conditions, including cells inside a 3D matrix, cell subjected to shear flows and biochemical stimuli, and cells in a live animal. Ballistic injection lasts <1 min and is followed by overnight incubation. Multiple particle tracking for one cell lasts <1 min. Forty cells can be examined in <1 h.

Figure 1

Minimal cell death is induced by ballistic injection of nanoparticles into the cytoplasm of adherent cells. (a–j) As a way of assessing whether ballistic injection induced cell death, we used fluorescence microscopy to measure both cell densities after 6 h of plating time (starting from the same plating density) (a) and the fractions of dead cells before ballistic injection (b–d) and after ballistic injection (e–g). We found that ballistic injection changed neither cell density (a) nor the extent of cell death compared with control cells that were not subjected to injection (b–g). In panel a, bars are the numbers of cells per unit area before ballistic injection and after ballistic injection for two independent trials (n = 2). (h–j) As a positive control, we found that the fraction of dead cells subjected to the nonionic surfactant Triton X-100 was effectively 100% (i,j). Nuclear DNA was stained using H33342 (blue in panels b,e and h); cell death was assessed using propidium iodide (red in panels c,f and i). Propidium iodide signal is only colocalized with nuclear DNA within the nucleus of dead cells. Scale bar, 100 µm. BI, ballistic injection.

Figure 2

Biolistic machine used to introduce submicron particles inside cells for high-throughout ballistic nanorheology (htBIN) analysis. A high concentration of nanoparticles is placed on the microcarrier. A pressure drop is applied and nanoparticles are pushed through a wire mesh stopping screen. Use of this machine is described in detail in the text.

Figure 3

Image processing steps taken to enhance the high-resolution tracking of fluorescent nanoparticles in the cytoplasm of live cells. Steps 33–50 are detailed in the text. a.u., arbitrary units.

Figure 4

htBIN analysis of normal and cancer human ovarian epithelial cells. (a,b) Twenty-second-long trajectory (a) and corresponding MSD (b) of a ballistically injected, 100-nm-diameter, fluorescent, polystyrene nanoparticle embedded in the cytoplasm of an OSE10 normal human ovarian epithelial cell. The trajectory is color coded to show the evolution of the movements of the nanoparticle. (c) Ensemble-averaged MSDs of nanoparticles in OSE10 cells (blue line) and OVCAR3 human ovarian cancer cells (green line). At least 100 nanoparticles were tracked for each type of cell. (d) Averaged creep compliance, Γ(τ), of OSE10 (blue) and OVCAR3 cells (green) directly computed from c. (e) Frequency-dependent viscous (continuous line) and elastic moduli (threaded line), G′(ω) and G″(ω), of OSE10 cells (blue) and OVCAR3 (green) computed from ensemble-averaged MSDs in c. (f,g). Shear viscosity (f) and elastic modulus evaluated at a frequency ω = 1 s⁻¹ (g) of OSE10 cells (blue) and OVCAR3 (green). 1 Poise = 0.1 Pa.s; 1 dyn cm⁻² = 0.1 Pa.