Simultaneous Nanorheometry and Nanothermometry Using Intracellular Diamond Quantum Sensors

Abstract

The viscoelasticity of the cytoplasm plays a critical role in cell morphology, cell division, and intracellular transport. Viscoelasticity is also interconnected with other biophysical properties, such as temperature, which is known to influence cellular bioenergetics. Probing the connections between intracellular temperature and cytoplasmic viscoelasticity provides an exciting opportunity for the study of biological phenomena, such as metabolism and disease progression. The small length scales and transient nature of changes in these parameters combined with their complex interdependencies pose a challenge for biosensing tools, which are often limited to a single readout modality. Here, we present a dual-mode quantum sensor capable of performing simultaneous nanoscale thermometry and rheometry in dynamic cellular environments. We use nitrogen-vacancy centers in diamond nanocrystals as biocompatible sensors for in vitro measurements. We combine subdiffraction resolution single-particle tracking in a fluidic environment with optically detected magnetic resonance spectroscopy to perform simultaneous sensing of viscoelasticity and temperature. We use our sensor to demonstrate probing of the temperature-dependent viscoelasticity in complex media at the nanoscale. We then investigate the interplay between intracellular forces and the cytoplasmic rheology in live cells. Finally, we identify different rheological regimes and reveal evidence of active trafficking and details of the nanoscale viscoelasticity of the cytoplasm.

Keywords: biosensing; nanodiamond; nitrogen-vacancy center; quantum sensing; rheometry; single particle tracking; thermometry.

Figure 1

Diamond-based nanothermometer and nanorheometer. (a) An illustration of the cross-section of a cell grown on a custom sensing chip, consisting of a resistive temperature detector (blue), two resistive heaters (red), and a coplanar waveguide (green), which is used for accurate temperature control and microwave delivery. Inset: A nanodiamond interacts with its complex surroundings in the cytoplasm, including the microtubules (blue), actin filaments (pink), and mitochondria (yellow in background). (b) Real-time tracking is achieved by collecting the PL from a nanodiamond at two axially offset planes separated by 100 nm (red) as the excitation laser (green) orbits the last inferred position of the nanodiamond with a radius of 50 nm. Corrections (δ) in the transverse and axial directions are made to counteract any imbalance in PL along the orbit (indicated by the intensity of the red orbit) and between the top and bottom imaging planes (gray shaded planes). (c) An example trajectory of a nanodiamond undergoing Brownian motion in glycerol. The background grid has a spacing of 1 μm. (d) The transition frequencies of the NV ground state are temperature dependent and probed using ODMR (top right), with the central frequency of the ODMR spectrum decreasing with increasing temperature (blue to red).

Figure 2

Accuracy and precision of the nanodiamond nanothermometer and nanorheometer. (a) The substrate temperature (gray curve) is stepped by 4 °C every 15 min, with the corresponding temperature reported by NV ODMR (red data). (b) The frequency shift is proportional to the change in temperature, with a temperature dependence of κ = −60.0 ± 0.4 kHz/°C. (c) The temperature precision over an accumulation time is characterized by the Allan deviation, from which we extract a sensitivity of . (d) Comparison between the known position (cyan data) in the x-direction of a nanodiamond moved in a Brownian motion-manner and the tracker-reported position (blue data), with the corresponding difference (δ x) shown in the lower panel. The set diffusion coefficient is 2 × 10³ nm²/s for this measurement. (e) The measured diffusion coefficient using the mean square displacement (MSD) at a time interval of 1 s shows a close agreement with the input diffusion coefficient. (f) The dynamic tracking accuracy, which is the standard deviation of the discrepancy between the tracker and particle trajectory, depends on the diffusion coefficient. When the particle is stationary, our system has a benchmark spatial resolution of 3.7 nm with 9.6 ms update rate (black dashed curve).

Figure 3

Temperature and rheology measurements in abiotic media. (a) An example of the nanodiamond trajectory projected onto the transverse plane over 96 s in glycerol. Scale bar: 1 μm. (b) The diffusion coefficient measured at different temperature values (red) with a linear fit (solid black curve) from which a hydrodynamic radius of 28 ± 1 nm is extracted. The gray dashed line shows the temperature dependence of the diffusion coefficient assuming a fixed viscosity of 0.919 Pa s corresponding to glycerol at 21 °C. (c, d) The simultaneous determination of temperature (red circles) and viscosity (blue circles) in glycerol, a purely viscous medium, measured using a single nanodiamond which was tracked for 30 min. The gray curve in (c) shows the temperature read out by the sensing chip and (d) shows the corresponding diffusion coefficient using the radius extracted from (b). (e, f) The mean square displacement (MSD) and viscous (G′′) and elastic (G′) moduli in a viscoelastic medium, glycerol-cross-linked xanthan (GCX), at T_C = 28.7 °C (blue circles) and T_H = 39.3 °C (red circles) obtained from nanodiamond tracking. (g) Temperature dependence of G′ and G′′ at f = 2.7 Hz for alternating temperatures T_C (blue shaded) and T_H (red shaded) as measured by the sensing chip (gray curve). (e and f) are calculated from the first 3 min of data at T_C and T_H in (g) (first blue and first red shaded regions).Data for panels a–d stem from one nanodiamond, data for panels e–g from a different single nanodiamond.

Figure 4

Nanodiamond multimodal sensing in live cells. (a, b) Simultaneous readout of temperature and power spectral density (PSD) in a cell. The gray curve in (a) shows the temperature read out by the sensing chip, and the PSD in (b) corresponds to f = 40 Hz. The dashed line represents the upper bound of the thermal contribution to the PSD. (c) Trajectory of a nanodiamond in a cell over 40 min xyz scale bar: 250 nm. Inset: xy particle trajectory relative to the optical diffraction limit (black spot, diameter = 500 nm). (d) xy particle trajectory showing both nondirected (dark blue) and directed (light blue) motion. Scale bar = 500 nm. (e) The mean square displacement, MSD, and ensemble averages (thick lines) for nanodiamonds with nondirected motion (blue) and directed motion (red) in untreated cells, and motion in cells treated with 50 μ M nocodazole for 1 h (gray). (f) Probability densities for the power-law exponents, α, for directed motion (red) and nondirected motion (blue) in untreated cells and cells that had been treated with 50 μM nocodazole for 1 h (gray). The black curves show fitted normal distributions.