## 4 Discussion  
Information entropy or homogeneity changes in an inspected medium can reflect the motion of particles within that medium. For optimal sensitivity of these measures, the right length- and time-scales should be chosen for inspection, such that they suit the particle size and expected motion. To illustrate this principle, we first provided examples of particle motion in homogeneous and non-homogeneous media. Theoretical analysis of a non-homogeneous medium suggests ways to improve the characterization of live cells under typical experimental conditions. Specifically, we considered parameters such as the pixel size, the size of the particles under study, the number of pixels used in analysis, the particle diffusivity and the time-lag between acquisitions. Evaluation of the motion of large intracellular particles (such as vesicles or organelles) is particularly important for study of the cell physiology. These large particles are embedded within the actin cytoskeletal mesh, and, accordingly, their motion reflects the motion of the cell cytoskeleton and the expenditure of intracellular mechanical work. As shown, fluctuations in the information entropy of selected pixels can reflect dynamics of the inspected medium in live cells. An important and inherent advantage of GLIE analysis is its sensitivity to intracellular mechanical work, which increases the homogeneity of the cell medium. In a more active and dynamic cell, the partition of the cell's constituents is reduced [6; 33], and so is the heterogeneity of the pixel gray levels and the average value of GLIE (Figure 8). Lower average GLIE values enable augmentation of momentary GLIE variation due to the logarithmic GLIE function (in Figure 3, the derivatives of GLIE curve are negatively correlated to GLIE values). This contributes to higher GLIE fluctuations in mechanically active cells. The application of ASD analysis to GLIE fluctuation, with the aid of power-fit parameters, enables the characterization of the cell's dynamic, including its constituents of diffusion coefficient, degree of anomalous diffusion and mechanical work. Cellular dynamics is affected by both passive factors (as elasticity or viscosity) and active ATP-dependent intracellular mechanical work. A sensitive tool to evaluate intracellular dynamics, along with its different contributing factors, should be very useful to study important cellular pathophysiological conditions. Such conditions may relate to energy imbalance and change of intracellular rheological factors. Cell conditions that effect ATP metabolism as cellular ischemia [37] or mitochondrial dysfunction [4; 8] were found to reduce intracellular mechanical work and intracellular dynamics. Mitochondrial dysfunction states relate to a wide area of clinical abnormalities from neurodegenerative diseases (like Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease) [38; 39], aging [40], autism [41], chronic fatigue syndrome [42], cancer [43] and skin diseases [44] to insulin resistance [45] and more. Accordingly, it is important to acquire a sensitive biophysical tool which will enable the detailed characterization of these influential pathophysiological conditions. In our results, we further demonstrated the ability of GLIE fluctuation parameters to distinguish a cellular state of mitochondrial dysfunction and ATP depletion. GLIE fluctuations parameters \(a,b,SSE\) and \(SA\) were all significantly higher in the normal active cells, as compared to the results in these cells after ATP depletion (Figure 6b). For validation of these results, we further analyzed the dynamics of these cells by using SICS fluctuation analysis (Figure 6a). As expected, the results from these two methods were correlated. Still, the GLIE fluctuations analysis method provided significantly more discriminating power for differentiating these two cellular metabolic states (Figure 7d). Malignant cells have high extent of intracellular mechanical work accompanied with low values of intracellular elasticity and viscosity [4].