Cells are highly dynamic systems that continuously undergo internal reconfiguration through random and/or coordinated molecular and mechanical responses [1
]. Intracellular dynamics such as the transport of molecules and cargos, along with cytoskeleton rearrangements, are fundamental processes that support a broad range of functions such as cell migration and division. These intracellular processes are derived from, and often influence physiological conditions of the cells. Malfunction of molecular motors, for instance, is believed to be responsible for incorrect distribution of chromosomes, one of the main signatures of Down syndrome [2
], and aberrant dynamics of the actin cytoskeleton is a distinct characteristic of invasive and metastatic cancer cells [3
]. Quantitative measurement of intracellular processes would thus aid in building a better understanding of the underlying mechanisms of cellular states and functions.
Conventional methods for quantifying structural dynamics in living cells have typically involved time course measurements of exogenous tracers conjugated to cellular structures [4
]. By monitoring the position of the tracers, these techniques probe molecular and mechanical responses such as cytoskeleton rearrangement. However, the introduction of exogenous tracers is likely to perturb the processes of interest, and the limited lifetime and uncertainties in the connectivity of the tracers to the structures may hinder a reliable characterization of cellular processes at various timescales [7
Dynamic light scattering (DLS) is a widespread optical technique used to measure the diffusive properties of scattering particles in suspension [8
]. Owing to its non-invasiveness and high sensitivity to particle size distribution, DLS has extensively been utilized to study protein kinetics [10
], biopolymer aggregation [12
], and for environmental sensing [13
]. DLS has been combined with microscopes to study the diffusive properties of biological and material specimens with high spatial resolution [14
]. Yet, these methods to date have been based only on scattered light intensity, incapable of measuring directional transport dynamics with nanometer-level sensitivity.
Recent advances in quantitative phase microscopy techniques enabled accurate measurement of amplitude and phase of optical waves related to sample structures and dynamics [19
]. The combination of these phase imaging techniques with field-based light scattering spectroscopy has been demonstrated, and used to examine structural changes of cells and tissues by measuring scattering signatures as a function of scattering angles [23
]. While those methods allow for assessing the heterogeneity of the sample structures, the measurement of time-dependent diffusive and directional dynamics of biological specimens such as living cells have not been demonstrated.
Here, we describe microscopic field-based dynamic light scattering (F-DLS) as a label-free, non-invasive technique to measure localized diffusive and directional transport dynamics. F-DLS makes full use of the amplitude and phase of optical waves scattered from structures inside a microscopic probe volume. Temporal autocorrelation analysis on the complex-valued signals enables to obtain mean-squared displacement (MSD) and time-averaged displacement (TAD) of scattering structures. The MSD represents the time-averaged variance of scatterer displacements, which gives access to diffusive properties of samples such as diffusion coefficients. The TAD, on the other hand, offers a statistical means to measure directional transport dynamics of scattering structures. Combination of F-DLS with a high-resolution microscope therefore leads to a novel detection method capable of measuring dynamic features of biological specimens with high spatial resolution.
We have performed theoretical and numerical analysis on F-DLS measurement, as detailed in Ref [26
]. As such, in this manuscript, we focus mainly on experimental verification and applications of F-DLS. We show the validity of F-DLS analysis based on the measurement of emulsion particles, and demonstrate F-DLS as a tool for characterizing localized intracellular dynamics inside human ovarian cancer cells (OVCAR-5s). We non-perturbatively observe the transition from random to directional intracellular processes on a timescale of ~0.01 sec, which have been reported and confirmed in other systems. The physiological importance of the directional processes on a timescale of 1~5 sec is further examined by monitoring their disruptions for Colchicine-treated and ATP-depleted cells. This study demonstrates the potential of the F-DLS technique in characterizing mechanical properties of dynamic systems, which ultimately could impact a broad range of biomedical applications.