Diffusive and directional intracellular dynamics measured by field-based dynamic light scattering

Abstract

Quantitative measurement of diffusive and directional processes of intracellular structures is not only critical in understanding cell mechanics and functions, but also has many applications, such as investigation of cellular responses to therapeutic agents. We introduce a label-free optical technique that allows non-perturbative characterization of localized intracellular dynamics. The method combines a field-based dynamic light scattering analysis with a confocal interferometric microscope to provide a statistical measure of the diffusive and directional motion of scattering structures inside a microscopic probe volume. To demonstrate the potential of this technique, we examined the localized intracellular dynamics in human epithelial ovarian cancer cells. We observed the distinctive temporal regimes of intracellular dynamics, which transitions from random to directional processes on a timescale of ~0.01 sec. In addition, we observed disrupted directional processes on the timescale of 1 approximately 5 sec by the application of a microtubule polymerization inhibitor, Colchicine, and ATP depletion.

Fig. 1

Top: SD-OCPM detection scheme. SD-OCPM uses the light back-reflected from the bottom surface of a coverslip as a reference to ensure the phase stability in measuring amplitude and phase of sample light scattered from the focal volume. Bottom: A broadband light source illuminates a fiber-based common-path interferometer. The light coupled to the sample arm is delivered to a specimen via an integrated laser-scanning inverted microscope. The backscattered light is re-coupled to the fiber for the subsequent interference spectrum measurement at the detection arm.

Fig. 2

(a) Measured amplitude and phase fluctuation of the interference between the reflected light from the top and bottom surfaces of a coverslip. The phase stability was measured as ~~9.8 × 10⁻³ rad at the measured SNR of ~37.5 dB. (b-d) Magnitude of the autocorrelation function, MSD, and TAD. The high stability of SD-OCPM produced a flat correlation with a value of ~1. The mean MSD and TAD were found as ~4.5 × 10⁻⁷ μm² and ~5.4 × 10⁻⁹ μm, respectively.

Fig. 3

F-DLS measurement for emulsion particles undergoing Brownian (black dot) and directional (green and blue dots) motion. The volume flow rates of 80 μL/min and 160 μL/min were used for the flow experiments. (a) SD-OCPM depth-resolved intensity distribution. The peak indicated by the red dot represents the interference between the light scattered from the emulsion particles inside the focal volume and the light reflected from the bottom surface of a coverslip. (b) Magnitudes of complex autocorrelation functions for the emulsion particles in Brownian and directional motion. The correlation function for the higher volume flow rate exhibits a shorter time constant. (c) MSDs: The power-law fits to MSDs found the exponents as ~1.04 for the particles in Brownian motion, and as ~1.07 and ~1.10 for the flow cases, respectively. The measured diffusion coefficient for the particles in Brownian motion (~0.99 μm²/sec) agreed with the predicted value (~1.07 μm²/sec) to within ~7%. (d) TADs calculated by the phase information of the complex autocorrelation functions. The static measurement exhibited no net time-averaged displacement, while the flow measurements showed linear behaviors with average velocities of –6.4 μm/sec (Q = 80 μL/min) and –12.5 μm/sec (Q = 160 μL/min), which agreed to the predicted values to within ~16% and ~20%, respectively.

Fig. 4

F-DLS applied to monitoring intracellular dynamics of OVCAR-5 cells. (a) Representative amplitude image of the cells at a depth of ~3.4 μm above the top surface of the collagen-I coated coverslip. Highly scattering intracellular structures such as mitochondria and cytoskeleton may account for the observed image contrast. The scale bar denotes 10 μm. (b) Measured amplitude and phase fluctuation recorded at the position indicated in (a). (c-d) Summary of F-DLS analysis: averages of log-transformed MSDs and magnitudes of TADs (N = 62). The intracellular dynamics is shown to vary dramatically from random to directional processes at ~0.01 sec. The inset in (d) is the log-log plot of TAD for better visualization of the short timescale. The thickening of the average TAD profile is due to the large standard errors on the long timescales. (e-f) Averages of log-transformed MSDs and the magnitudes of TADs for the fixed cells (N = 47). The fixed cells exhibit significantly different diffusive characteristics with no directional transport. The error bar represents ± SE, and is included in (c-f).

Fig. 5

Summarized F-DLS results for control (N = 62), Colchicine-treated (N = 57) and ATP-depleted (N = 68) OVCAR-5 cells. (a) Averages of log-transformed MSDs. (b-c) The mean exponents on the short timescale did not show a remarkable difference (*: p<0.5), but on the long timescale, the mean exponents for the control, Colchicine and ATP-depleted cells were 0.73, 0.54, and 0.38, respectively (****: p<0.0005). The error bar represents ± SE. (d) TADs: Most control cells exhibited directed motion as evident from the linear TAD profiles, whereas the Colchicine-treated and ATP-depleted cells showed randomized behaviors. (e) Velocity correlation coefficient as a function of time-delay, Δτ, averaged over all the measurements in each condition. The shorter time constants for Colchicine-treated and ATP-depleted cells indicate significant disruption of directional intracellular dynamics. The correlation diagrams at Δτ = 0.5 sec (inset) demonstrate the transition from correlated to randomized motion for Colchicine-treated and ATP-depleted cells.